CN118139880A

CN118139880A - O-linked N-acetylglucosamine hydrolase (OGA) inhibitor combination therapy

Info

Publication number: CN118139880A
Application number: CN202280070979.2A
Authority: CN
Inventors: K·比格兰; A·S·弗莱舍; D·J·梅戈特; H·N·纳托尔
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 2021-10-22
Filing date: 2022-10-21
Publication date: 2024-06-04
Also published as: CA3235104A1; IL312045A; AU2022371475A1; WO2023070064A1

Abstract

The present invention provides methods of treating and/or preventing and/or slowing the progression of diseases characterized by amyloid β deposition and/or diseases characterized by amyloid β deposition and abnormal tau aggregation. Such methods comprise administering to a patient in need of such treatment an effective amount of an anti-N3 pG aβ antibody and an effective amount of an OGA inhibitor.

Description

O-linked N-acetylglucosamine hydrolase (OGA) inhibitor combination therapy

The present disclosure relates to combinations of one or more O-linked N-acetylglucosamine hydrolase (O-GlcNAcase, "OGA") inhibitors and one or more anti-N3 pGlu amyloid β (anti-N3 pgΑβ) antibodies, and methods of using the same to treat disorders characterized by a combination of i) amyloid β (Αβ) deposition and/or ii) amyloid β (Αβ) deposition and tau-mediated neurodegeneration. Some aspects of the disclosure relate to treating Alzheimer's Disease (AD).

Alzheimer's disease is a devastating neurodegenerative disease characterized pathologically by amyloid beta deposition and/or abnormal tau aggregation. AD affects millions of people worldwide, and the treatment of AD is one of the most important unmet needs of society. The neuropathological hallmark of AD is the presence of intracellular neurofibrillary tangles containing hyperphosphorylated tau protein. Another pathological hallmark of AD is the presence of amyloid β (aβ) deposition. Interactions between tau protein and aβ pathology are still under interpretation. Aβ may trigger tau pathology, and a more complex and synergistic interaction between aβ and tau may be shown later, and drive disease progression (Busche et al Nature Neuroscience 23:1183-93 (2020)).

An OGA inhibitor of brain penetration is expected to provide a treatment for tau-mediated neurodegenerative disorders such as alzheimer's disease. Antibodies that target aβ (e.g., anti-N3 pGlu aβ antibodies) have shown promise for the removal of amyloid deposits in the brain of individuals and are used/developed as therapeutics for alzheimer's disease. The present disclosure provides i) certain compounds as OGA inhibitors in combination with anti-N3 pGlu aβ antibodies, and ii) related methods of treating diseases mediated by aberrant tau and/or amyloid deposition (e.g., AD).

Microtubule-associated protein tau oligomerizes into a filamentous structure, such as Paired Helical Filaments (PHF) and straight or twisted filaments, producing neurofibrillary tangles (NFT) and neurofibrillary filaments (NT), one of the typical pathological features of alzheimer's disease and other tauopathies (tauopathies). The number of NFTs in the brain of an individual with alzheimer's disease is closely related to the severity of the disease. This suggests that tau has a key role in neuronal dysfunction and neurodegeneration (Nelson et al J Neuropathol Exp neurol.,71 (5), 362-381 (2012)). Tau pathology has been shown to be associated with the course of PSP; cases with more severe progression have higher tau loads than those with slower progression (Williams et al, brain,130,1566-76 (2007)). Recent studies (Yuzwa et al, nat Chem Biol,4 (8), 483-490 (2008)) support the therapeutic potential of O-GLCNACASE (OGA) inhibitors to limit tau protein hyperphosphorylation and aggregation to pathological tau for the treatment of Alzheimer's disease and related tau protein mediated neurodegenerative disorders. In particular, OGA inhibitors Thiamet-G are associated with slowing motor neuron loss in JNPL tau mouse models (Yuzwa et al, nat Chem Biol,8,393-399 (2012)) and with reduction of tau pathology and dystrophic neurites in Tg4510 tau mouse models (Graham et al, neuropharmacology,79,307-313 (2014)). Thus, OGA inhibitors are considered to be effective therapeutic approaches to reduce accumulation of tau hyperphosphorylation pathological forms (e.g., NFT and NT). U.S. patent No. 9,120,781 discloses hexahydrobenzoxazole and hexahydrobenzothiazole derivatives having OGA inhibitory activity and further discloses that they are useful in the treatment of diseases and disorders associated with OGA deficiency or overexpression and/or accumulation or deficiency of 2-acetamido-2-deoxy-5 beta-D-glucopyranoside (O-GlcNAc). Furthermore, US2016/0031871 discloses certain glycosidase inhibitors for the treatment of alzheimer's disease.

The accumulation of amyloid β peptide in the form of brain amyloid deposition is an early and important event in alzheimer's disease, leading to neurodegeneration and thus to onset of clinical symptoms such as cognitive and dysfunction ((Selkoe, JAMA 283:1615-7 (2000); hardy et al, science 297:353-6 (2002), masters et al, nat.Rev.Dis.Primers 1:15056 (2015), and Selkoe et al, EMBO mol.Med.8:595-608 (2016)), amyloid beta is an integral membrane protein formed by proteolytic cleavage of a larger glycoprotein called Amyloid Precursor Protein (APP), which is expressed in many tissues, especially in neuronal synapses, is cleaved by gamma secretase, liberates Aβ peptides, wherein the Aβ monomers comprise a set of peptides of 37-49 amino acid residues that aggregate into various types of higher structures, including oligomers, fibrils and amyloid fibrils, the amyloid oligomers are soluble and may diffuse to the entire brain, while amyloid fibrils are larger and insoluble and may further aggregate to form amyloid deposits or a mixture of amyloid deposits found in human patients, including the Aβ peptide(s) that are also found in human patients, including pG 3. Beta peptide(s) and the N-terminal end of pG.3. Beta peptide (pG.beta.3-beta peptide) is also found in the heterogeneous form of pG.3. Beta peptide (p-beta.42, p-beta peptide, p-beta.p-beta peptide is found, and are present only in amyloid deposits. N3pGlu A.beta.lacks the first two amino acid residues at the N-terminus of human A.beta.and has a pyroglutamic acid derived from glutamic acid at the third amino acid position of A.beta.s. Although the N3pGlu aβ peptide is a minor component of the deposition aβ in the brain, studies have shown that the N3pGlu aβ peptide has positive aggregation properties and accumulates early in the deposition cascade.

Antibodies to N3pGlu aβ are known in the art. For example, U.S. patent No. 8,679,498; U.S. patent No. 8,961,972; U.S. patent No. 10,647,759; and U.S. patent No. 11,078,261 (incorporated herein by reference in its entirety) discloses anti-N3 pGluA beta antibodies, methods of making antibodies, antibody formulations, and methods of treating diseases such as alzheimer's disease with such antibodies.

In various animal models, passive immunization with antibodies against aβ (including N3pGlu aβ) has been demonstrated to disrupt aβ aggregation and promote clearance of deposits in the brain by chronic administration of antibodies over a long period of time. Donanemab (disclosed in U.S. Pat. No. 8,679,498) is a pyroglutamic acid-modified antibody directed against the third amino acid of the amyloid β (N3 pGlu aβ) epitope present only in brain amyloid deposits.

Donanemab therapeutic and prophylactic strategies include targeting amyloid deposition specific N3pGlu aβ in a population of early symptomatic AD patients with brain amyloid loading. The theoretical basis is based on the amyloid hypothesis of AD, which suggests that aβ production and deposition are early and essential events in the pathogenesis of AD. See, e.g., selkoe, JAMA 283:1615-1617 (2000), the entire contents of which are incorporated herein by reference. Donanemab have recently shown efficacy/potency in removing amyloid deposits and slowing the progression of AD. See, e.g., mintun et al, NEW ENGLAND Journal of Medicine, 384.18:1691-1704 (2021), the entire contents of which are incorporated herein by reference.

The combination of antibodies that specifically bind to anti-N3 pG aβ and reduce amyloid β in the brain of a human individual with an OGA inhibitor is expected to provide treatment for diseases such as AD. This combination may reduce pathogenic tau species and tau aggregates and reduce amyloid beta (aβ). Such a combination may also preferably be more efficient than either molecule alone. For example, treatment with such a combination may allow for the use of lower doses of either or both molecules, potentially resulting in lower side effects (or shorter duration of treatment of one or the other) than each molecule alone, while maintaining efficacy. It is believed that the combinations provided herein will not only reduce amyloid β, but also reduce abnormal tau, tau aggregation into pathological tau and its transmission, for use in the treatment of diseases such as AD.

In one aspect, the disclosure is directed to a method of treating a patient suffering from a disorder characterized by amyloid β (aβ) deposition. In some embodiments, the disclosure relates to methods of treating a patient suffering from a disease characterized by amyloid β deposition and abnormal tau aggregation. The method comprises administering to a patient in need thereof an effective amount of an anti-N3 pG aβ antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of the formula:

Or a pharmaceutically acceptable salt thereof. In some embodiments, the methyl group at the 2-position of the OGA inhibitor is in the cis configuration relative to the oxygen at the 4-position of the piperidine ring:

In some embodiments, the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide. In some embodiments, the OGA inhibitor is crystalline. In some embodiments, N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1- [ piperidinyl ] methyl ] thiazol-2-yl ] acetamide is characterized by a combination of a peak having a diffraction angle 2- θ of 12.1 ° with one or more peaks selected from 15.3 °, 21.6 °, 22.2 °, 22.7 °, 23.5 °, 24.3 °, and 26.8 ° in an X-ray powder diffraction spectrum, with a diffraction angle error of 0.2 °. Another aspect of the present disclosure relates to a method of treating a patient suffering from a disorder characterized by amyloid β (aβ) deposition, the method comprising administering to the patient in need thereof an effective amount of an anti-N3 pG aβ antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of the formula:

Or a pharmaceutically acceptable salt thereof. In some embodiments, the disclosure relates to methods of treating a patient suffering from a disease characterized by amyloid β deposition and abnormal tau aggregation. In some embodiments, the methyl group at the 2-position of the OGA inhibitor is in the cis configuration relative to the oxygen at the 4-position of the piperidine ring:

In some embodiments, the methyl group at the 2-position of the OGA inhibitor is in the trans configuration relative to the oxygen at the 4-position of the piperidine ring:

In some embodiments, the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide. In some embodiments, the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide. In some embodiments, the OGA inhibitor is crystalline. In some embodiments, the OGA inhibitor is characterized by an X-ray powder diffraction spectrum having a peak with a diffraction angle 2- θ of 13.5 ° combined with one or more peaks selected from 5.8 °, 13.0 °, 14.3 °, 17.5 °, 20.4 °, 21.4 °, and 22.2 °, with a diffraction angle error of 0.2 °.

Another aspect of the present disclosure relates to a method of treating a patient suffering from a disorder characterized by amyloid β (aβ) deposition, the method comprising administering to the patient in need thereof an effective amount of an anti-N3 pG aβ antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of the formula:

Or a pharmaceutically acceptable salt thereof. In some embodiments, the disclosure relates to methods of treating a patient suffering from a disease characterized by amyloid β deposition and abnormal tau aggregation. In some embodiments, the methyl group at position 5 of the OGA inhibitor is in the cis configuration relative to the oxygen at position 3 of the piperidine ring:

in some embodiments, the methyl group at position 5 of the OGA inhibitor is in the trans configuration relative to the oxygen at position 3 of the piperidine ring:

In some embodiments, the OGA inhibitor is 1- (2- (((3 r,5 s) -1- ((6-fluoro-2-methylbenzo [ d ] thiazol-5-yl) methyl) -5-methylpyrrolidin-3-yl) oxy) -5, 7-dihydro-6H-pyrrolo [3,4-b ] pyridin-6-yl) ethan-1-one. In some embodiments, the OGA inhibitor is crystalline.

Yet another aspect of the present disclosure relates to a method of treating a patient suffering from a disease characterized by amyloid β deposition and/or abnormal tau aggregation, comprising administering to the patient in need thereof an effective amount of an anti-N3 pG aβ antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of the formula:

In some embodiments, the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide. In some embodiments, the OGA inhibitor is crystalline. In some embodiments, N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1- [ piperidinyl ] methyl ] thiazol-2-yl ] acetamide is characterized by a combination of a peak having a diffraction angle 2- θ of 12.1 ° with one or more peaks selected from 15.3 °, 21.6 °, 22.2 °, 22.7 °, 23.5 °, 24.3 °, and 26.8 ° in an X-ray powder diffraction spectrum, with a diffraction angle error of 0.2 °.

Yet another aspect of the present disclosure relates to a method of treating a patient suffering from a disease characterized by amyloid β deposition and/or abnormal tau aggregation, comprising administering to a patient in need of such treatment an effective amount of an anti-N3 pG aβ antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of the formula:

In some embodiments, the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide. In some embodiments, the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide. In some embodiments, the OGA inhibitor is crystalline. In some embodiments, the OGA inhibitor is characterized by a combination of a peak having a diffraction angle 2- θ of 13.5 ° and one or more peaks selected from 5.8 °, 13.0 °, 14.3 °, 17.5 °, 20.4 °, 21.4 °, and 22.2 ° in the X-ray powder diffraction spectrum, with a diffraction angle error of 0.2 °.

or a pharmaceutically acceptable salt thereof. In some embodiments, the methyl group at position 5 of the OGA inhibitor is in the cis configuration relative to the oxygen at position 3 of the piperidine ring:

The invention also provides a method of treating a cognitive or neurodegenerative disease comprising administering to a patient in need thereof a treatment comprising an effective amount of an anti-N3 pGlu aβ antibody in combination with an effective amount of an OGA inhibitor. The invention also provides a method of treating clinical or preclinical AD comprising administering to a patient in need thereof a treatment comprising an effective amount of an anti-N3 pGlu aβ antibody in combination with an effective amount of an OGA inhibitor. The invention also provides a method of treating prodromal AD (sometimes also referred to as mild cognitive impairment or MCI), mild AD dementia, moderate AD dementia and/or severe AD dementia, comprising administering to a patient in need thereof a treatment comprising an effective amount of an anti-N3 pGlu aβ antibody in combination with an effective amount of an OGA inhibitor.

In some embodiments, the present disclosure provides a method of treating, preventing, or slowing the functional/cognitive decline in a patient diagnosed with preclinical Alzheimer's Disease (AD) (also known as pre-symptomatic AD), clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, severe AD dementia, down's syndrome, clinical cerebral amyloid angiopathy, or preclinical cerebral amyloid angiopathy. Such methods comprise administering to a patient in need of such treatment an effective amount of an anti-N3 pG aβ antibody in combination with an effective amount of an OGA inhibitor disclosed herein. The invention also provides a method of preventing memory loss, cognitive decline or functional decline in a clinically asymptomatic individual with low or very low levels of aβ1-42 in cerebrospinal fluid (CSF) and/or low or very low aβ deposition in the brain, comprising administering an effective amount of an anti-N3 pG aβ antibody in combination with an effective amount of an OGA inhibitor as disclosed herein. In some embodiments, clinically asymptomatic individuals are known to have a genetic mutation that causes alzheimer's disease. In the present disclosure, "clinically asymptomatic individuals known to have a genetic mutation to cause Alzheimer's disease" includes patients known to have a genetic mutation to cause Alzheimer's disease (Paisa mutation, a genetic mutation that causes autosomal dominant Alzheimer's disease), or patients at higher risk of developing AD due to carrying one or two APOE 4 alleles. In some embodiments, the present disclosure provides a method of treating, preventing or slowing cognitive/functional decline in a patient known to have a PSEN 1E 280A-induced alzheimer's disease genetic mutation (Paisa mutation, a genetic mutation that causes autosomal dominant alzheimer's disease) or a patient carrying one or two APOE 4 alleles comprising administering to a patient in need of such treatment an effective amount of an anti-N3 pga beta antibody in combination with an effective amount of an OGA inhibitor disclosed herein.

In some embodiments, the present disclosure also provides a method of treating, preventing, or slowing cognitive/functional decline in a patient diagnosed with preclinical Alzheimer's Disease (AD), clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, and severe AD dementia, comprising administering to a patient in need of such treatment an effective amount of an anti-N3 pgaβ antibody in combination with an effective amount of an OGA inhibitor disclosed herein. The invention also provides a method of preventing memory loss or cognitive/functional decline in a clinically asymptomatic patient with low NFT levels and/or low amyloid deposition levels in the brain comprising administering an effective amount of an anti-N3 pG aβ antibody in combination with an effective amount of an OGA inhibitor disclosed herein.

Another embodiment of the invention provides a method for preventing progression of mild cognitive impairment to AD comprising administering to a patient in need of such treatment an effective amount of an anti-N3 pgaβ antibody in combination with an effective amount of an OGA inhibitor.

This embodiment also provides an antibody against N3pG aβ for simultaneous, separate or sequential use in combination with an OGA inhibitor for treatment.

The invention further provides a pharmaceutical composition comprising an anti-N3 pG aβ antibody and one or more pharmaceutically acceptable carriers, diluents or excipients in combination with a pharmaceutical composition comprising an OGA inhibitor and one or more pharmaceutically acceptable carriers, diluents or excipients.

In addition, the invention provides a kit comprising an anti-N3 pG aβ antibody and an OGA inhibitor. The invention further provides a kit comprising a pharmaceutical composition comprising an anti-N3 pG aβ antibody (with one or more pharmaceutically acceptable carriers, diluents, or excipients) and a pharmaceutical composition comprising an OGA inhibitor (with one or more pharmaceutically acceptable carriers, diluents, or excipients). As used herein, a "kit" includes separate containers containing individual components in a single package, wherein one component is an anti-N3 pG aβ antibody and the other component is an OGA inhibitor. The "kit" may also comprise separate containers of individual components in separate packages, one of which is an anti-N3 pG aβ antibody and the other is an OGA inhibitor, with instructions for administration of the individual components as a combination.

The invention also provides the use of an anti-N3 pG aβ antibody in the manufacture of a medicament for the treatment of AD, mild AD, prodromal AD or for the prevention of progression of mild cognitive impairment to AD, wherein the medicament is for simultaneous, separate or sequential administration with an OGA inhibitor.

In some embodiments of the disclosure, the anti-N3 pgaβ antibody comprises a Heavy Chain (HC) and a Light Chain (LC), wherein HC comprises a Heavy Chain Variable Region (HCVR) and LC comprises a Light Chain Variable Region (LCVR), said HCVR comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3, and said LCVR comprises CDRs LCDR1, LCDR2 and LCDR3. In some embodiments, the anti-N3 pga β antibodies of the invention have the amino acid sequence of LCDR1 given by SEQ ID No.5, the amino acid sequence of LCDR2 given by SEQ ID No.6, the amino acid sequence of LCDR3 given by SEQ ID No.7, the amino acid sequence of HCDR1 given by SEQ ID No.8, the amino acid sequence of HCDR2 given by SEQ ID No.9, and the amino acid sequence of HCDR3 given by SEQ ID No. 10.

In one embodiment, the invention provides an anti-N3 pG aβ antibody comprising a LCVR and a HCVR, wherein the amino acid sequence of the LCVR is given by SEQ ID No.1 and the amino acid sequence of the HCVR is given by SEQ ID No. 2. In a further embodiment, the invention provides an anti-N3 pG aβ antibody comprising a Light Chain (LC) and a Heavy Chain (HC), wherein the amino acid sequence of LC is given by SEQ ID No.3 and the amino acid sequence of HC is given by SEQ ID No. 4.

In some embodiments, the anti-N3 pga β antibodies of the invention have the amino acid sequence of LCDR1 given by SEQ ID No.15, the amino acid sequence of LCDR2 given by SEQ ID No.16, the amino acid sequence of LCDR3 given by SEQ ID No.17, the amino acid sequence of HCDR1 given by SEQ ID No.18, the amino acid sequence of HCDR2 given by SEQ ID No.19, and the amino acid sequence of HCDR3 given by SEQ ID No. 20. In one embodiment, the invention provides an anti-N3 pG aβ antibody comprising a LCVR and a HCVR, wherein the amino acid sequence of the LCVR is given by SEQ ID No.11 and the amino acid sequence of the HCVR is given by SEQ ID No. 12. In another embodiment, the invention provides an anti-N3 pG aβ antibody comprising a Light Chain (LC) and a Heavy Chain (HC), wherein the amino acid sequence of LC is given by SEQ ID No.13 and the amino acid sequence of HC is given by SEQ ID No. 14.

The anti-N3 pG aβ antibodies of the invention may be prepared and purified using known methods. For example, cDNA sequences encoding HC of anti-N3 pGAβ antibodies and cDNA sequences encoding LC of anti-N3 pGAβ antibodies can be cloned and engineered into GS (glutamine synthetase) expression vectors. The engineered immunoglobulin expression vector may then be stably transfected into CHO cells. Those skilled in the art will appreciate that expression of mammalian antibodies will result in glycosylation, typically at highly conserved N-glycosylation sites in the Fc region. It can be verified whether the stable clone expresses an antibody that specifically binds to amyloid deposition or N3pG aβ. Positive clones can be expanded into serum-free medium for antibody production in a bioreactor. The antibody-secreting medium may be purified by conventional techniques. For example, the medium can be conveniently applied to a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer (e.g.phosphate buffered saline). The column may be washed to remove non-specific binding members. The bound antibodies may be eluted, for example, by a pH gradient, and the antibody fractions may be detected using techniques such as by SDS-PAGE, and subsequently pooled. The antibodies can be concentrated and/or sterile filtered using conventional techniques. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion, hydrophobic interactions, ion exchange or hydroxyapatite chromatography. The product may be frozen immediately, for example at-70 ℃, or may be lyophilized.

The anti-N3 pga beta antibody of the invention binds to human N3 pga beta (also referred to as N3pGlu a beta). In one embodiment, the anti-N3 pG aβ antibodies of the invention bind to conformational epitopes of human N3pG aβ.

As used herein, an "antibody" is an immunoglobulin molecule comprising two Heavy Chains (HC) and two Light Chains (LC) interconnected by disulfide bonds. The amino-terminal portion of each LC and HC includes a variable region responsible for antigen recognition through the Complementarity Determining Regions (CDRs) contained therein. CDRs are interspersed with regions that are more conserved, called framework regions. The assignment of amino acids within the LCVR and HCVR regions of the antibodies of the invention to CDR domains is based on the following: kabat numbering convention (Kabat et al, ann.N.Y. Acad.Sci.190:382-93 (1971); kabat et al ,Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242(1991)), and North numbering convention (North et al ,A New Clustering of Antibody CDR Loop Conformations,Journal of Molecular Biology,406:228-256(2011)). determined the CDRs of the antibodies of the invention as described above).

The anti-N3 pGlu Abeta antibodies of the invention include kappa LC and IgG HC. In a specific embodiment, the anti-N3 pGlu aβ antibody of the invention is a human IgG1 isotype.

The antibodies of the invention are monoclonal antibodies ("mabs"). Monoclonal antibodies can be produced, for example, by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques such as CDR grafting, or combinations of such techniques, or by other techniques known in the art. The monoclonal antibodies of the invention are human or humanized. Humanized antibodies may be engineered to comprise one or more human framework regions (or substantially human framework regions) surrounding CDRs derived from a non-human antibody. Human framework germline sequences can be obtained from ImMunoGeneTics through their website imgtObtained or obtained from Marie-Paule Lefranc and GERARD LEFRANC, handbook of immunoglobulin profiles (Immunoglobulin FactsBook, academic25Press,2001,ISBN 012441351). In another embodiment of the invention, the antibody or nucleic acid encoding the antibody is provided in isolated form. As used herein, the term "isolated" refers to a protein, peptide, or nucleic acid that is not found in nature and is free or substantially free of other macromolecular species found in the cellular environment. As used herein, "substantially free" means that the protein, peptide or nucleic acid of interest comprises more than 80% (by mole), preferably more than 90%, and more preferably more than 95% of the macromolecular species present.

The anti-N3 pGlu aβ antibodies of the invention or their combination with an OGA inhibitor are administered in the form of a pharmaceutical composition. The pharmaceutical compositions of the invention may be administered by parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular) to patients at risk of, or exhibiting, the diseases or disorders described herein. Subcutaneous and intravenous routes are preferred. In some embodiments, the pharmaceutical compositions of the invention are administered by intravenous infusion.

The terms "treatment", "treatment" or "to treatment" and the like include inhibiting, slowing or stopping the progression or severity of an existing symptom, condition, disease or disorder in a patient. The term "patient" or "individual" refers to a human.

The term "preventing" refers to the prophylactic administration of an antibody of the invention to an asymptomatic patient or to a patient suffering from preclinical alzheimer's disease to prevent the onset or progression of the disease.

The term "a disease characterized by aβ deposition (deposition)" or "a disease characterized by aβ deposition (deposits)" is used interchangeably and refers to a disease characterized pathologically by aβ deposition in the brain or in the brain vasculature of an individual. This includes Alzheimer's Disease (AD), down's Syndrome (DS), and Cerebral Amyloid Angiopathy (CAA). Clinical diagnosis, staging or progression of Alzheimer's disease can be readily determined by an attending diagnostician or health care professional as a person skilled in the art by using known techniques and by observation. This typically includes some form of brain plaque imaging, mental or Cognitive assessment (e.g., clinical dementia assessment-block abstracts (CLINICAL DEMENTIA RATING-surcharge of boxes, CDR-SB), easy mental state examination (Mini-MENTAL STATE Exam, MMSE) or Alzheimer's disease assessment scale (Alzheimer' S DISEASE ASSESSMENT SCALE-cognition, ADAS-Cog) or functional assessment (e.g., alzheimer's disease partnership studies-activities of daily living (ADCS-ADL). Cognitive and functional assessment may be used to determine changes in cognition (e.g., cognitive decline) and changes in function (e.g., functional decline) in a patient as used herein are stages of established Alzheimer's disease.

In some embodiments, an individual is positive for amyloid deposition when amyloid is detected in the brain by methods such as amyloid imaging with radiolabeled PET compounds or using diagnostic agents that detect aβ or aβ biomarkers. Exemplary methods that may be used in the present disclosure to measure brain amyloid load include, for example, flurbiproflumilast (Florbetapir) (Carpenter et al ,"The Use of the Exploratory IND in the Evaluation and Development of¹⁸F-PET Radiopharmaceuticals for Amyloid Imaging in the Brain:A Review of One Company's Experience,"The Quarterly Journal of Nuclear Medicine and Molecular Imaging 53.4:387(2009),, the entire contents of which are incorporated herein by reference); and flumetanol (Flutemetamol) (Heurling et al ,"Imagingβ-amyloid Using[¹⁸F]Flutemetamol Positron Emission Tomography:From Dosimetry to Clinical Diagnosis,"European Journal of Nuclear Medicine and Molecular Imaging 43.2:362-373(2016),, the entire contents of which are incorporated herein by reference).

[ ¹⁸ F ] -flurobeta-pir allows for qualitative and quantitative measurement of brain plaque load in patients, including those suffering from pre-AD or mild AD dementia. For example, the absence of a significant [ ¹⁸ F ] -flurobeta pizide signal in the visual reading indicates that patients clinically exhibiting cognitive impairment have sparse or even no amyloid plaques. Thus, [ ¹⁸ F ] -flurobeta piride also provides confirmation of amyloid pathology. [ ¹⁸ F ] -flurobetaziram PET also provides a quantitative assessment of fibrillar amyloid plaques in the brain, and in some embodiments, can be used to assess the reduction of amyloid plaques in the brain caused by the antibodies of the present disclosure.

Amyloid imaging with radiolabeled PET compounds can also be used to determine whether aβ deposition in the brain of a human patient is reduced or increased (e.g., calculating the percent reduction in aβ deposition after treatment or assessing the progression of AD). The normalized uptake value ratio (SUVr) values obtained from amyloid imaging (with radiolabeled PET compound) can be correlated by one skilled in the art to calculate the% reduction in aβ deposition in the patient's brain before and after treatment. SUVr values can be converted to normalized centiloid units, where 100 is the average of AD and 0 is the average of young controls, allowing comparison between amyloid PET tracers and calculation of the reduction from centiloid units (Klunk et al ,"The Centiloid Project:Standardizing Quantitative Amyloid Plaque Estimation by PET,"Alzheimer's&Dementia 11.1:1-15(2015) and Navitsky et al ,"Standardization of Amyloid Quantitation with Florbetapir Standardized Uptake Value Ratios to the Centiloid Scale,"Alzheimer's&Dementia 14.12:1565-1571(2018),, the entire contents of which are incorporated herein by reference). In some embodiments, the change in brain amyloid plaque deposition from baseline is measured by [ ¹⁸ F ] -fluroxypyr PET scan.

Amyloid load can also be measured for the purposes of this disclosure using a cerebrospinal fluid or plasma based beta-amyloid assay. For example, aβ42 can be used to measure cerebral amyloid (Palmqvist, s. Et al ,"Accuracy of Brain Amyloid Detection in Clinical Practice Using Cerebrospinal Fluid Beta-amyloid 42:a Cross-validation Study Against Amyloid Positron Emission Tomography.JAMA Neurol 71,1282-1289(2014),, the entire contents of which are incorporated herein by reference). In some embodiments, the ratio of aβ42/aβ40 or aβ42/aβ38 may be used as a biomarker for amyloid β (Janelidze et al ,"CSF Abeta42/Abeta40 and Abeta42/Abeta38 Ratios:Better Diagnostic Markers of Alzheimer Disease,"Ann Clin Transl Neurol 3,154-165(2016),, the entire contents of which are incorporated herein by reference).

In some embodiments, the deposited brain amyloid plaques or aβ in CSF or plasma can be used to group individuals and identify which group of individuals respond to treatment/prevention of a disease (as described herein) using the antibodies or methods described herein.

Tau levels in the brain of a human individual can be determined using methods such as Tau imaging with radiolabeled PET compounds (Leuzy et al ,"Diagnostic Performance of RO948 F18 Tau Positron Emission Tomography in the Differentiation of Alzheimer Disease from Other Neurodegenerative Disorders,"JAMA Neurology 77.8:955-965(2020);Ossenkoppele et al ,"Discriminative Accuracy of[¹⁸F]-flortaucipir Positron Emission Tomography for Alzheimer Disease vs Other Neurodegenerative Disorders,"JAMA 320,1151-1162,(2018),, the entire contents of which are incorporated herein by reference).

In some embodiments, the biomarker [ ¹⁸ F ] -florbtaucipir as a PET ligand may be used for the purposes of the present disclosure. PET tau images can be quantitatively assessed, for example, by the disclosed methods (Pontecorvo et al ,"AMulticentre Longitudinal Study of Flortaucipir(¹⁸F)in Normal Ageing,Mild Cognitive Impairment and Alzheimer's Disease Dementia,"Brain 142:1723-35(2019);Devous et al ,"Test–Retest Reproducibility for the Tau PET Imaging Agent Flortaucipir F18,"Journal of Nuclear Medicine 59:937-43(2018);Southekal et al ,"Flortaucipir F18 Quantitation Using Parametric Estimation of Reference Signal Intensity,"J.Nucl.Med.59:944-51(2018),, the entire contents of which are incorporated herein by reference), to estimate SUVr (normalized uptake value ratio) and/or visually assess a patient, for example, to determine whether the patient has AD pattern (Fleisher et al ,"Positron Emission Tomography Imaging With[¹⁸F]-flortaucipir and Postmortem Assessment of Alzheimer Disease Neuropathologic Changes,"JAMA Neurology 77:829-39(2020),, the entire contents of which are incorporated herein by reference). A lower SUVr value indicates less tau load, while a higher SUVr value indicates higher tau load. In one embodiment, the quantitative assessment by flortaucipir scans is accomplished by an automated image processing pathway, as described in Southekal et al ,"Flortaucipir F18 Quantitation Using Parametric Estimation of Reference Signal Intensity,"J.Nucl.Med.59:944–951(2018), which is incorporated herein by reference in its entirety. In some embodiments, counts within specific targeted regions of interest in the brain (e.g., multi-block barycentric discriminant analysis or MUBADA, see Devous et al ,"Test-Retest Reproducibility for the Tau PET Imaging Agent Flortaucipir F18,"J.Nucl.Med.59:937–943(2018),, incorporated herein by reference in its entirety) are compared to reference regions, such as the entire cerebellum (wholeCere), cerebellum GM (cereCrus), map-based white matter (atlasWM), individual-specific WM (ssWM, e.g., parameter estimation using reference signal intensity (PERSI), see Southekal et al ,"Flortaucipir F18Quantitation Using Parametric Estimation of Reference Signal Intensity,"J.Nucl.Med.59:944–951(2018),, incorporated herein by reference in its entirety). The preferred method of determining tau load is a quantitative analysis reported as a normalized uptake value ratio (SUVr) that represents counts (e.g., MUBADA) within a particular targeted region of interest in the brain compared to a reference region (e.g., using PERSI).

In some embodiments, phosphorylated tau (P-tau; phosphorylated at threonine 181 or 217) may be used to measure tau load/burden for purposes of this disclosure (Barthelemy et al ,"Cerebrospinal Fluid Phospho-tau T217 Outperforms T181 as a Biomarker for the Differential Diagnosis of Alzheimer's Disease and PET Amyloid-positive Patient Identification,"Alzheimer's Res.Ther.12,26(2020);Mattsson et al ,"AβDeposition is Associated with Increases in Soluble and Phosphorylated Tau that Precede a Positive Tau PET in Alzheimer's Disease,"Science Advances 6(16),(2020);, the entire contents of which are incorporated herein by reference). In a specific embodiment, for purposes of this disclosure, antibodies to threonine phosphorylated human tau at residue 217 may be used to measure tau load/burden in an individual (see international patent application publication No. WO 2020/242963, the entire contents of which are incorporated by reference). In some embodiments, the disclosure includes using the anti-tau antibodies disclosed in WO 2020/242963 to measure tau load in an individual. The anti-tau antibodies disclosed in WO 2020/242963 are directed against human tau isoforms expressed in the CNS (e.g., recognize isoforms expressed in the CNS and not recognize human tau isoforms expressed only outside the CNS). Such antibodies against human tau isoforms expressed in the CNS are useful in methods of identifying/selecting patients as one or more of: (i) suffering from the diseases disclosed herein; (ii) at risk of having a disease disclosed herein; (iii) in need of treatment for the diseases disclosed herein; or (iv) a need for neuroimaging.

The reduction or alleviation of cognitive decline may be measured by cognitive assessment such as simple mental state examination (MMSE) or Alzheimer's disease assessment scale (ADAS-Cog). The reduction or alleviation of functional decline may be measured by functional assessment such as Alzheimer's disease collaborative research-activities of daily living (ADCS-ADL). The reduction or alleviation of function and cognitive decline may be measured by integrated measures such as clinical dementia assessment-block abstract (CDR-SB) or Integrated Alzheimer's Disease Rating Scale (iADRS).

As used herein, "mg/kg" refers to the amount of milligrams of antibody or drug administered to a patient based on the patient's body weight (in kilograms). One dose is administered at a time. For example, for a patient weighing 70kg, a10 mg/kg dose of antibody would be a single 700mg dose of antibody administered in a single administration. For a patient weighing 70kg, the 20mg/kg dose of antibody will be a single 1400mg dose of antibody administered in a single administration. Similarly, for a patient weighing 80kg, the 40mg/kg dose of antibody would be the 3200mg dose of antibody administered in a single administration.

As used herein, the phrase "in combination" refers to the administration of an anti-N3 pGlu aβ antibody of the invention simultaneously or sequentially in any order with another molecule (a "combination molecule", e.g., an OGA inhibitor, symptomatic drug, or aβ antibody), or in any combination thereof. In some embodiments, the phrase "in combination" refers to the simultaneous or sequential administration of an anti-N3 pGlu aβ antibody of the invention with an OGA inhibitor, or in any combination thereof. The two molecules may be administered as part of the same pharmaceutical composition or in separate pharmaceutical compositions. The anti-N3 pGlu aβ antibody may be administered prior to, concurrently with, or after the administration of the OGA inhibitor, or in some combination of these ways. When the combination is administered at repeated intervals (e.g., during standard treatment), the anti-N3 pGlu aβ antibody may be administered prior to, concurrently with, or after each administration of the OGA inhibitor, or in some combination of these, or at different intervals associated with treatment with the OGA inhibitor, or at a single dose or series of doses prior to, during, or after the course of treatment with the OGA inhibitor.

In particular embodiments of the invention, antibodies and antibody fragments (e.g., anti-N3 pG aβ antibodies) or nucleic acids encoding the same may be provided in isolated form. As used herein, the term "isolated" refers to a protein, peptide, or nucleic acid that is not found in nature and is free or substantially free of other macromolecular species found in the cellular environment. As used herein, "substantially free" means that the protein, peptide or nucleic acid of interest comprises more than 80% (by mole), preferably more than 90%, and more preferably more than 95% of the macromolecular species present.

In some embodiments, the antibodies of the invention are expressed in cell culture. After expression and/or secretion of the antibodies and antibody fragments of the invention, the medium is clarified to remove cells and the clarified medium is purified using any of a number of commonly used techniques. Purified antibodies and antibody fragments can be formulated into pharmaceutical compositions according to well known methods for formulating proteins and antibodies for parenteral administration, particularly for subcutaneous, intrathecal or intravenous administration. The antibodies and antibody fragments may be lyophilized with suitable pharmaceutically acceptable excipients and subsequently reconstituted with a water-based diluent prior to use. Alternatively, the antibodies and antibody fragments may be formulated in aqueous solution and stored prior to use. In either case, the storage and injection forms of the pharmaceutical compositions of the antibodies and antibody fragments will contain pharmaceutically acceptable excipient or excipients, which are ingredients other than the antibodies and antibody fragments. Whether an ingredient is pharmaceutically acceptable depends on its effect on safety and efficacy or on the safety, purity and efficacy of the pharmaceutical composition. An ingredient is pharmaceutically unacceptable for use in pharmaceutical compositions for antibodies and antibody fragments if it is judged to have a sufficiently adverse effect on safety or effectiveness (or on safety, purity or efficacy) that it is reasonably considered to be unusable in the composition for administration to humans.

The novel combinations and methods of the invention include brain penetration OGA inhibitors. In some embodiments of the novel combinations and methods of the invention, the OGA inhibitor comprises a compound of formula I:

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the OGA inhibitors of the novel combinations and methods of the invention are compounds of formula Ia:

Or a pharmaceutically acceptable salt thereof.

Certain configurations of formula I, which comprise embodiments of the OGA inhibitors of the novel combinations and methods of the invention, further comprise:

And pharmaceutically acceptable salts thereof.

5-Methyl-1, 2, 4-oxadiazol-3-yl compounds of formula I wherein the methyl and oxygen substituents on the piperidine ring are in either the cis or trans configuration, or pharmaceutically acceptable salts thereof, are included within the scope of the novel combination OGA inhibitors of the present invention. The novel combinations of the present invention also contemplate all individual enantiomers and diastereomers, as well as mixtures of enantiomers, including racemates, of the 5-methyl-1, 2, 4-oxadiazol-3-yl compounds of the present invention. The absolute configurations of 5-methyl-1, 2, 4-oxadiazol-3-yl compounds of the novel combinations and methods provided herein include:

N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide and pharmaceutically acceptable salts thereof; and

N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide is particularly preferred.

The 5-methyl-1, 2, 4-oxadiazol-3-yl OGA inhibitor compounds or salts thereof of the present invention can be prepared by a variety of methods known to those of ordinary skill in the art. Those of ordinary skill in the art recognize that the specific synthetic steps of each pathway described may be combined in different ways, or with steps from different schemes, to prepare the compounds of the invention or salts thereof. The products of the following steps may be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents are as previously defined unless otherwise indicated. Reagents and starting materials are readily available to those of ordinary skill in the art. Without limiting the scope of the invention, the following preparation methods and examples are provided to further illustrate the invention. In addition, it is understood by those of ordinary skill in the art that compounds of formulas Ia, ib, ic and Id may be prepared by using starting materials having the corresponding stereochemical configuration which may be prepared by those of ordinary skill in the art. For example, the following preparation examples use starting materials having a configuration that ultimately corresponds to formula Ia.

The following preparations and examples are intended to illustrate but not limit the invention and various modifications may be made by those of ordinary skill in the art. The following examples and assays demonstrate that the anti-N3 pG aβ antibodies of the invention are useful in the treatment of diseases characterized by aβ deposition, such as alzheimer's disease, down's syndrome, and CAA.

Preparation example 1: synthesis of tert-butyl N- (4-fluoro-5-formyl-thiazol-2-yl) carbamate.

Cesium fluoride (227 g,1480 mmol) is added to a solution of tert-butyl N- (4-chloro-5-formyl-thiazol-2-yl) carbamate (38.8 g,148mmol; see, e.g., N.Masuda et al, bioorg Med Chem,12,6171-6182 (2004)) in dimethyl sulfoxide (DMSO, 776 mL) for preparation of tert-butyl N- (4-chloro-5-formyl-thiazol-2-yl) carbamate at room temperature. The reaction mixture was stirred in a 145 ℃ heating block for 48 hours at an internal temperature of 133 ℃ and the mixture was cooled in an ice water bath. To the mixture were added saturated aqueous sodium bicarbonate (500 mL), saturated aqueous NaCl (500 mL), and ethyl acetate (500 mL). The mixture was stirred at room temperature for 10 min, filtered through celite, and washed with ethyl acetate (500 mL). The filtrate was transferred to a separatory funnel and the layers were separated, and then the aqueous layer was extracted with ethyl acetate (1L). The combined organics were washed with saturated aqueous NaCl solution (1L) and then the saturated aqueous NaCl solution layer was extracted with ethyl acetate (300 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated to give a residue. The residue was passed through a pad of silica gel (330 g), eluted with 5% ethyl acetate in dichloromethane (1.5L), and the filtrate was concentrated under reduced pressure to give a residue (24.2 g).

The residue (combined batch 32.7g,133 mmol) was dissolved in isopropanol (303 mL), filtered and purified by SFC (supercritical fluid chromatography) using an IC column (cellulose polysaccharide derivative: tris (3, 5-dichlorophenyl carbamate, 30X 250mm,5 μm) with 10% isopropanol (no additive) at 180 mL/min, 3mL of the sample size. The fractions containing the product were concentrated to give the title compound (16.1 g,48% yield.) MS M/z 247.0 (M+H).

Preparation example 2: synthesis of N- (4-fluoro-5-formyl-thiazol-2-yl) acetamide (method A).

Zinc bromide (91.9 g,408 mmol) was added in one portion to a mixture of tert-butyl N- (4-fluoro-5-formyl-thiazol-2-yl) carbamate (33.5 g,136 mmol) and methylene chloride (503 mL) at room temperature in a jacketed vessel. The reaction mixture was stirred at an internal temperature of 37 ℃ overnight, then the jacket temperature was set to-10 ℃ and tetrahydrofuran (111 mL) was added dropwise over 15 minutes, keeping the internal temperature below 6 ℃. The jacket temperature was then set to-30℃and pyridine (110 mL,1360 mmol) was added dropwise over 5 minutes, keeping the internal temperature below 5 ℃. The jacket temperature was set to 0deg.C and acetic anhydride (116 mL,1220 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred at 37 ℃ internal temperature overnight, cooled to room temperature, and eluted with tetrahydrofuran (500 mL) through a short pad of celite. The filtrate was transferred to a flask, and the mixture was concentrated under reduced pressure to give a residue, which was concentrated again from toluene (50 mL). To the residue was added a solution of citric acid monohydrate (57.2 g,272 mmol) in water (400 mL) and 2-methyltetrahydrofuran (400 mL), and the mixture was stirred at 40 ℃ for 5 minutes. The mixture was passed through a short pad of celite eluting with 2-methyltetrahydrofuran (100 mL). The filtrate was transferred to a separatory funnel and the layers were separated. The aqueous layer was extracted with 2-methyltetrahydrofuran (2X 250 mL) and the combined organic extracts were diluted with water (500 mL). Solid sodium bicarbonate was added to the mixture in portions over 5 minutes while stirring until gas evolution ceased. The mixture was transferred to a separatory funnel and the layers were separated; the aqueous layer was extracted with 2-methyltetrahydrofuran (200 mL and 100 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue, which was diluted with 2-methyltetrahydrofuran (100 mL), and the mixture was eluted with 2-methyltetrahydrofuran (2.5L) through a short pad of silica gel (250 g). The filtrate was concentrated under reduced pressure to give a residue, which was suspended in a 1:1 mixture of dichloromethane and heptane (202 mL). The mixture was stirred at room temperature for 30 minutes and filtered. The filtered solid was collected and dried in vacuo at 40 ℃ for 2 hours to give the title compound (18.0 g,70% yield). MS M/z189.0 (M+H).

Alternative synthesis of N- (4-fluoro-5-formyl-thiazol-2-yl) acetamide (method B).

Dichloromethane (1325 g,15.6 mol) was added to 2-amino-4-chlorothiazole-5-carbaldehyde (100 g,0.61 mol) and pyridine (194.6 g,2.46 mol) and cooled to 0-5 ℃. Acetic anhydride (188.4 g,1.85 mol) was added dropwise, maintaining the temperature at 0-5 ℃. After the addition was completed, the temperature was adjusted to 20-25 ℃ and stirred for 41 hours. Concentrated under reduced pressure, followed by the addition of 35% aqueous hcl (200 mL) and water (1.5L) maintaining the temperature below 40 ℃. Cooled to 20-25 ℃ and stirred for 18 hours. The resulting mixture was filtered and the collected solids were washed with water. The solid was dried at 60-65 ℃ for 24 hours to give N- (4-chloro-5-formylthiazol-2-yl) acetamide (75 g,66% yield).

Sulfolane (1000 mL) was added to N- (4-chloro-5-formylthiazol-2-yl) acetamide (50 g,0.24mol, prepared directly above), tetramethylammonium chloride (107.1 g,0.98 mol) and cesium fluoride (370.6 g,2.4 mol) under an inert atmosphere. Heat to 130 ℃ and stir for 23 hours. HPLC analysis showed 75% conversion of the title compound in 45% in situ.

Alternative synthesis of N- (4-fluoro-5-formyl-thiazol-2-yl) acetamide (method C).

2-Propanol (150 mL) was added to tetramethyl ammonium fluoride tetrahydrate (10.2 g,109.0 mmol) and the mixture was concentrated to about half volume (internal temperature maintained at 70 ℃ C.) under vacuum to remove water. 2-propanol (200 mL) was added and the mixture was concentrated to about half volume under vacuum. And repeated twice more. Dimethylformamide (DMF, 200 mL) was added and concentrated to about half volume in vacuo. Tetrahydrofuran (THF, 200 mL) was added and concentrated to about half volume. And repeated twice more. N- (4-chloro-5-formylthiazol-2-yl) acetamide (1.22 g,6.0mmol, prepared in method B above) and DMF (12 mL) were added. Heated to 110 ℃ and stirred for 12 hours. The reaction mixture was cooled to 25 ℃. 2-methyltetrahydrofuran (40 mL) and water (40 mL) were added. The resulting layer was separated and the aqueous layer was extracted with 2-methyltetrahydrofuran (40 mL). The layers were separated and the combined organic layers were washed with water (20 mL). The layers were separated and the organic layer was concentrated under reduced pressure. Ethyl acetate (20 mL) and water (5 mL) were added, the resulting layers were separated, and the organic layer was concentrated to remove the solvent. Ethyl acetate (2 mL) and heptane (2 mL) were added, and the resulting mixture was filtered. The filtered solid was dried under vacuum at 55 ℃ for 18 hours to give the title compound as a 93% mixture with N- (4-chloro-5-formylthiazol-2-yl) acetamide.

Preparation example 3: synthesis of (2S, 4S) -4-hydroxy-2-methyl-piperidine-1-carboxylic acid tert-butyl ester.

To the flask was added (2S) -2-methyl-4-oxo-piperidine-1-carboxylic acid tert-butyl ester (50 g,234.4 mmol) and tetrahydrofuran (500 mL). The mixture was cooled to-65 ℃ under nitrogen and 1M solution of lithium tris (sec-butyl) borohydride in tetrahydrofuran (304.77 ml,304.8 mmol) was added dropwise over 45 minutes, keeping the internal temperature below-60 ℃. The reaction mixture was stirred at room temperature for 1 hour and cooled to-30 ℃. To the reaction mixture was added a mixture of water (25.3 mL) and tetrahydrofuran (100.2 mL), keeping the internal temperature below-20 ℃. An aqueous solution of 30% w/w hydrogen peroxide (118.9 mL,1.2 mol) in water (126.70 mL) was added dropwise over 1 hour, keeping the internal temperature below 10 ℃. To the mixture was added 5M aqueous HCl (46.9 mL,234.4 mmol) and methyl tert-butyl ether (1L), and the mixture was warmed to room temperature. The layers were separated and the organic phase was stirred for 10 minutes at room temperature with a solution of sodium metabisulfite (222.8 g,1.17 mol) in water (500 mL). The layers were separated and the organic phase was dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 0-50% methyl tert-butyl ether/isohexane, combining the product containing fractions and concentrating under reduced pressure to give the title compound (40.4 g,78% yield). ES/MS M/z 238 (M+Na). Alternative synthesis of (2 s,4 s) -4-hydroxy-2-methyl-piperidine-1-carboxylic acid tert-butyl ester.

DMSO (27.4 kg,1.0 vol) and D- (+) -glucose monohydrate (28.9 kg,1.25 eq) were added to a glass-lined reactor containing deionized water (460L) and potassium dihydrogen phosphate (6.5 kg,0.41 eq) at 20deg.C. The internal temperature was adjusted to 30 ℃ and the pH of the reaction was adjusted to 6.9 by adding 8% aqueous sodium hydroxide (15 l,0.3 eq). To the reactor was added (2S) -2-methyl-4-oxo-piperidine-1-carboxylic acid tert-butyl ester (24.9 kg,1.0 eq (99.1% ee)), and the mixture was stirred at 30℃for 15 min. Ketoreductase (KRED-130, 250g,1% w/w), glucose dehydrogenase (GDH-101, 250g,1% w/w) and NADP sodium salt (63 g,0.25% w/w) were added directly to the reaction mixture through the opening. The mixture was maintained at a temperature of 30 ℃ and pH 7.0±0.2 by the addition of 8% aqueous nahco ₃. After stirring for 16.5 hours, celite (12.5 kg,50% w/w) and toluene (125 l,5 vol) were added to the reaction. After stirring at 30 ℃ for 30 minutes, the mixture was transferred to another 2000L reactor over 1 hour through a series GAF filter (4 sockets). The mixture was allowed to stand without stirring for 30 minutes, the layers were separated, and the aqueous layer was back-extracted with toluene (2×125L). The combined organic layers were filtered (series GAF filter) and the toluene mixture was washed with 25% aqueous sodium chloride (125 l,5 vol) at 25 ℃. The resulting toluene solution was azeotropically dried (partial vacuum, internal temperature <60 ℃) to 0.10 wt/wt% water and cooled to 20 ℃. The mixture was filtered from the reactor through a cartridge filter into a clean barrel under positive nitrogen pressure. The reaction mixture was then transferred from the barrel into a 500L glass lined vessel and concentrated under vacuum (< 60 ℃) to a target residual volume of about 56L (2.25 vol). N-heptane (169 kg,10 vol) was added at 40℃and 25g of (2S, 4S) -4-hydroxy-2-methylpiperidine-1-carboxylic acid tert-butyl ester was added to the mixture. The resulting viscous slurry was diluted with additional n-heptane (25L, 1 vol) and cooled to 16℃over 4 hours. The product was isolated by centrifugation, washed with n-heptane (25L each; 4 spin-drying is required) and dried in a tray dryer at 30℃for 11 hours to give 20.3kg (81% yield; >99.9% ee). ES/MS M/z 238 (M+Na).

Preparation example 4: synthesis of tert-butyl (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] piperidine-1-carboxylate.

3- (Chloromethyl) -5-methyl-1, 2, 4-oxadiazole (43.5 g,301 mmol) was added to a solution of (2S, 4S) -4-hydroxy-2-methyl-piperidine-1-carboxylic acid tert-butyl ester (29.5 g,137 mmol) in acetonitrile (590 mL) at room temperature. The reaction mixture was stirred in an ice water bath and sodium tert-butoxide (54.3 g, 268 mmol) was added in portions over 10 minutes keeping the internal temperature below 10 ℃. The reaction mixture was stirred in an ice-water bath for 9 hours (internal temperature 5 ℃), slowly warmed to room temperature, and stirred overnight. The reaction mixture was cooled in an ice-water bath and saturated aqueous ammonium chloride solution (200 mL) was added over 5 minutes, maintaining the internal temperature below 10 ℃ during the addition. The mixture was then diluted with water (100 mL) and warmed to room temperature. The mixture was extracted with methyl tert-butyl ether (2X 300 mL) and the combined organic extracts were washed with saturated aqueous sodium chloride (300 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated to give a residue. The residue was passed through a pad of silica gel (300 g), eluted with methyl tert-butyl ether (1L) and the filtrate was concentrated under reduced pressure to give the title compound (46.5 g, >99% yield). MS M/z 334.0 (M+Na).

Alternative synthesis of (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] piperidine-1-carboxylic acid tert-butyl ester.

To a solution of (2S, 4S) -4-hydroxy-2-methyl-piperidine-1-carboxylic acid tert-butyl ester (0.25 g,1.16 mmol) and 3- (chloromethyl) -5-methyl-1, 2, 4-oxadiazole (308 mg,2.3 mmol) in N, N-dimethylformamide (3 mL) was added sodium tert-butoxide (0.35 g,3.5 mmol) in portions over 5 minutes under nitrogen at 0deg.C. The reaction mixture was stirred at room temperature for 10 minutes and at 40 ℃ for 12 hours. The reaction mixture was cooled to room temperature and quenched with water (10 mL). The layers were separated and the aqueous phase was extracted with methyl tert-butyl ether (2X 10 mL). The combined organic extracts were washed with 5% aqueous lithium chloride, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the title compound (0.49 g,81% yield, 60% purity) as a brown oil. MS M/z 334.0 (M+Na).

Preparation example 5: synthesis of 5-methyl-3- [ [ (2S, 4S) -2-methyl-4-piperidinyl ] oxymethyl ] -1,2, 4-oxadiazole hydrochloride.

A flask containing (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] piperidine-1-carboxylic acid tert-butyl ester (4.0 g,12.9 mmol) was immersed in an ice-water bath. A solution of 4M hydrochloric acid in 1, 4-dioxane (25.9 mL,104 mmol) was added dropwise to the flask over 5 minutes with stirring, maintaining the internal temperature below 20℃during the addition. The reaction mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure to give the title compound (3.5 g, 92% yield based on 83% purity as measured by ¹ HNMR). MS M/z 212.0 (M+H).

Alternative synthesis of 5-methyl-3- [ [ (2S, 4S) -2-methyl-4-piperidinyl ] oxymethyl ] -1,2, 4-oxadiazole hydrochloride.

Methanol (50 mL) was added to (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] piperidine-1-carboxylic acid tert-butyl ester (12.9 g,0.04 mol). The mixture was cooled to 0 ℃. A solution of 4M hydrochloric acid in methanol (80 mL) was added dropwise to the cooled mixture, keeping the internal temperature below 20 ℃. The reaction mixture was stirred at room temperature for 18 hours. The mixture was concentrated to remove the solvent. Acetone (10 mL) was added and the mixture stirred for 20 min. Tetrahydrofuran (40 mL) was added and the mixture was stirred for 3 hours. The solids were collected by filtration under nitrogen and the filtered solid cake was rinsed with tetrahydrofuran. The filtered solid was dried in vacuo at 45 ℃ for 2 hours to give the title compound in 90% purity. Recrystallization using acetone can increase the purity of the title compound to 95%.

Preparation example 6: synthesis of 5-methyl-3- [ [ (2S, 4S) -2-methyl-4-piperidinyl ] oxymethyl ] -1,2, 4-oxadiazole.

To a solution of tert-butyl (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] piperidine-1-carboxylate (0.49 g,1.6 mmol) in dichloromethane (10 mL) under nitrogen was added trifluoroacetic acid (1.8 mL,23 mmol). The mixture was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure to give a yellow oil. The residue was dissolved in methanol (5 mL) and poured onto a cation exchange column eluting with methanol (2 x 10 mL) followed by a solution of 2M ammonia in methanol (10 mL). The filtrate was concentrated under reduced pressure to give the title compound (0.3 g,91% yield). MS M/z 212.0 (M+H).

In other embodiments of the novel combinations and methods of the invention, the OGA inhibitor is a compound of formula X:

Or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides compounds of formula Xa:

Or a pharmaceutically acceptable salt thereof.

Certain configurations of formula X, which comprise embodiments of the OGA inhibitors of the novel combinations and methods of the invention, further comprise:

And pharmaceutically acceptable salts thereof.

5-Methyl-1, 3, 4-oxadiazol-2-yl compounds of formula X wherein the methyl and oxygen substituents on the piperidine ring are in either the cis or trans configuration, or pharmaceutically acceptable salts thereof, are included within the scope of the novel combination of OGA inhibitors of the present invention. The novel combinations of the present invention also contemplate all individual enantiomers and diastereomers, as well as mixtures of enantiomers, including racemates, of the 5-methyl-1, 3, 4-oxadiazol-2-yl compounds of the present invention. The absolute configurations of 5-methyl-1, 3, 4-oxadiazol-2-yl compounds of the novel combinations and methods provided herein include:

N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide and pharmaceutically acceptable salts thereof, including the free base, and including crystalline forms.

The 5-methyl-1, 3, 4-oxadiazol-2-yl compounds or salts thereof of the novel combinations and methods of the invention can be prepared by a variety of methods known to those of ordinary skill in the art, some of which are illustrated in the schemes, preparations and examples below. Those of ordinary skill in the art recognize that the specific synthetic steps of each of the pathways described may be combined in different ways, or with steps from different schemes, to prepare the compounds of the invention or salts thereof. The products of the following steps may be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents are as previously defined unless otherwise indicated. Reagents and starting materials are readily available to those of ordinary skill in the art. Without limiting the scope of the invention, the following schemes, preparations and examples are provided to further illustrate the invention. In addition, it is understood by those of ordinary skill in the art that the compounds of formulas Xa, xb, xc and Xd can be prepared by using starting materials having the corresponding stereochemical configurations, which can be prepared by those of ordinary skill in the art. For example, the following preparation examples use starting materials having a configuration that ultimately corresponds to formula Xa.

Preparation example 7: synthesis of 2- [ [ (2S, 4S) -1-tert-butoxycarbonyl-2-methyl-4-piperidinyl ] oxy ] acetic acid.

2-Chloro-1-morpholino-ethanone (59.4 g, 803 mmol) was added to a solution of (2S, 4S) -4-hydroxy-2-methyl-piperidine-1-carboxylic acid tert-butyl ester (52.1 g,242 mmol) in acetonitrile (521 mL) at room temperature. The reaction mixture was stirred in an ice water bath and sodium tert-butoxide (48.0 g, 284 mmol) was added in portions over 10 minutes maintaining the internal temperature below 15 ℃. The reaction mixture was stirred at room temperature for 2 hours and added to another flask containing saturated aqueous ammonium chloride (250 mL) and water (250 mL) over 5 minutes while cooling with an ice water bath maintaining the internal temperature below 15 ℃ during the addition. The mixture was warmed to room temperature and extracted with methyl tert-butyl ether (2×500 mL), and the combined organic extracts were washed with saturated aqueous sodium chloride (300 mL). The combined organics were dried over sodium sulfate, filtered and concentrated to give a residue which was combined with 2-propanol (414 mL) and 2M aqueous sodium hydroxide (303 mL,605 mmol) at room temperature. The reaction mixture was stirred overnight in a 47 ℃ heating block at an internal temperature of 45 ℃. The reaction mixture was cooled to room temperature and concentrated to remove 2-propanol, and the mixture was diluted with water (50 mL). The mixture was extracted with methyl tert-butyl ether (250 mL) and the aqueous layer was cooled in an ice water bath and acidified with acetic acid (55.6 mL,968 mmol). The aqueous mixture was extracted with ethyl acetate (4X 250 mL); the combined organic extracts were dried over sodium sulfate and filtered; and concentrated to give a residue, which was concentrated from toluene (3×100 mL) to give the title compound (79.8 g, >99% yield). MS M/z 272.0 (M-H).

Preparation example 8: synthesis of (2S, 4S) -4- [2- (2-acetylhydrazino) -2-oxo-ethoxy ] -2-methyl-piperidine-1-carboxylic acid tert-butyl ester.

Tetrahydrofuran (798 mL) was added to a flask containing 2- [ [ (2S, 4S) -1-tert-butoxycarbonyl-2-methyl-4-piperidinyl ] oxy ] acetic acid (79.8 g,224 mmol) and the mixture was stirred in an ice-water bath (internal temperature 5 ℃). 1,1' -carbonyldiimidazole (43.5 g,268 mmol) was added in one portion to the mixture, and the reaction mixture was stirred at room temperature for 2 hours. Another part of 1,1' -carbonyldiimidazole (7.25 g,44.7 mmol) was added and the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was immersed in an ice-water bath and acetohydrazide (21.5 g,29 mmol) was added in one portion. The reaction mixture was stirred at room temperature overnight. The reaction mixture was cooled with stirring in an ice-water bath and saturated aqueous sodium bicarbonate solution (500 mL) was added over 2 minutes keeping the internal temperature below 15 ℃. The mixture was diluted with water (300 mL) and the resulting mixture was concentrated under reduced pressure to remove tetrahydrofuran. The resulting aqueous mixture was extracted with 2-methyltetrahydrofuran (4X 500 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue which was combined with ethyl acetate (200 mL) and heptane (200 mL). The mixture was stirred at room temperature for 30 minutes, diluted with heptane (200 mL), stirred vigorously at room temperature for another 30 minutes, and filtered. The filtered solid was dried in vacuo at 40 ℃ for 2 hours to give a first crop of the title compound (71.5 g). The filtrate was filtered again and the filtered solid was dried under a stream of nitrogen at room temperature for 15 minutes to give a second crop of the title compound (1.98 g). A major portion of the first (71.1 g,216 mmol) and second (1.97 g,5.98 mmol) were combined with tert-butyl methyl ether (731 mL) and the mixture was stirred in a 45℃heating block for 30 min at an internal temperature of 40℃cooled to room temperature over 1 hour with stirring and filtered. The filtered solid was dried under vacuum at room temperature under a nitrogen stream for 30 minutes to give the title compound (53.7 g,71% yield). MS M/z 352.0 (M+Na).

Preparation example 9: synthesis of tert-butyl (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] piperidine-1-carboxylate.

To a solution of (2S, 4S) -4-hydroxy-2-methyl-piperidine-1-carboxylic acid tert-butyl ester (0.5 g,2 mmol) in N, N-dimethylformamide (5 mL) was added sodium tert-butoxide (920 mg,9.3 mmol) in portions under nitrogen at room temperature. The resulting reaction mixture was stirred at room temperature for 40 minutes. The reaction mixture was cooled to 0deg.C and 2- (chloromethyl) -5-methyl-1, 3, 4-oxadiazole (416 mg,3.1 mmol) was added. The resulting solution was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and the resulting residue was diluted with water. The mixture was extracted with 3 parts of ethyl acetate. The combined organic extracts were dried over magnesium sulfate, filtered and concentrated under reduced pressure to give a crude oil.

The residue was dissolved in dimethyl sulfoxide (to a total volume of 2 mL) and purified by preparative HPLC (Phenomenex Gemini-NX 10Micron 30x100mm C-18) eluting with a gradient of 15% (CH ₃ CN & water (containing 10mM ammonium bicarbonate), adjusted with ammonium hydroxide to pH 9) to 100% CH ₃ CN for 7 min (50 mL/min, 1 sample injection, 204 nm) to give the title compound (28 mg,4% yield). MS M/z 312 (M+H).

Alternative synthesis of (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] piperidine-1-carboxylic acid tert-butyl ester.

To the flask were added (2 s,4 s) -4- [2- (2-acetylhydrazino) -2-oxo-ethoxy ] -2-methyl-piperidine-1-carboxylic acid tert-butyl ester (53.7 g,163 mmol) and acetonitrile (537 mL) and the slurry was stirred at room temperature. N, N-diisopropylethylamine (114 mL,652 mmol) and p-toluenesulfonyl chloride (77.7 g,408 mmol) were added in three portions to the mixture in one portion over 5 minutes with cooling in a water bath. The reaction mixture was stirred at room temperature overnight and then cooled in an ice-water bath. N ', N' -dimethylethane-1, 2-diamine (21.8 g,245 mmol) was added dropwise over 10 minutes keeping the internal temperature below 15 ℃. The reaction mixture was stirred at room temperature for 30 minutes, and diluted with saturated aqueous citric acid (50 mL), ethyl acetate (500 mL) and water (450 mL) at room temperature. The layers were separated and the organic layer was washed with a mixture of saturated aqueous citric acid (50 mL) and water (450 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (500 mL) and the aqueous layer was then extracted with ethyl acetate (500 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated to give a residue which was passed through a short pad of silica gel (400 g) eluting with 25% ethyl acetate in heptane (2X 500 mL) followed by ethyl acetate (5X 500 mL). The product-containing fractions were concentrated to give the title compound (53.3 g, >99% yield). MS M/z 312.2 (M+H).

Preparation example 10: synthesis of 5-methyl-5- [ [ (2S, 4S) -2-methyl-4-piperidinyl ] oxymethyl ] -1,3, 4-oxadiazole 2, 2-trifluoro acetic acid.

To a solution of tert-butyl (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] piperidine-1-carboxylate (27.5 mg,0.09 mmol) in dichloromethane (3 mL) under nitrogen was added trifluoroacetic acid (0.035 mL,0.45 mmol). The mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure to give the title compound (0.04 g,84% yield). MS M/z 212.0 (M+H).

Preparation example 11: synthesis of 2-methyl-5- [ [ (2S, 4S) -2-methyl-4-piperidinyl ] oxymethyl ] -1,3, 4-oxadiazole.

To the flask was added (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] piperidine-1-carboxylic acid tert-butyl ester (52.9 g,170 mmol) and dichloromethane (265 mL) at room temperature. The reaction mixture was stirred in an ice-water bath (internal temperature 5 ℃) and trifluoroacetic acid (265 mL,350 mmol) was added dropwise over 5 minutes keeping the internal temperature below 10 ℃. The reaction mixture was stirred at room temperature for 15min and concentrated to give a residue, which was diluted with water (300 mL) and methyl tert-butyl ether (300 mL). The layers were separated and the aqueous layer was stirred in an ice-water bath and basified with 50% aqueous sodium hydroxide (20 mL) maintaining the internal temperature below 10 ℃ during the addition. The mixture was extracted with dichloromethane (4×300 mL) and the combined organic extracts were dried over sodium sulfate, filtered and concentrated to give the title compound (30.5 g,85% yield). MS M/z212.2 (M+H).

In other embodiments of the novel combinations and methods of the invention, the OGA inhibitor is a compound of formula XX:

Or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides compounds of formula XXa:

Or a pharmaceutically acceptable salt thereof.

And pharmaceutically acceptable salts thereof.

5- (Methylpyrrolidin-3-yl) oxy compounds of formula XX, wherein the methyl and oxygen substituents on the pyrrolidine ring are in cis or trans configuration, or pharmaceutically acceptable salts thereof, are included within the scope of the novel combination OGA inhibitors of the present invention. The novel combinations of the present invention also contemplate all individual enantiomers and diastereomers, as well as mixtures of enantiomers, including racemates, of the 5- (methylpyrrolidin-3-yl) oxy compounds of the present invention. The absolute configuration of the 5- (methylpyrrolidin-3-yl) oxy compounds of the novel combinations and methods provided herein include:

1- (2- (((3R, 5S) -1- ((6-fluoro-2-methylbenzo [ d ] thiazol-5-yl) methyl) -5-methylpyrrolidin-3-yl) oxy) -5, 7-dihydro-6H-pyrrolo [3,4-b ] pyridin-6-yl) ethan-1-one, and pharmaceutically acceptable salts thereof, including the free base, and including crystalline forms.

The 5- (methylpyrrolidin-3-yl) oxy compounds or salts thereof of the novel combinations and methods of the invention may be prepared by a variety of methods known to those of ordinary skill in the art, some of which are illustrated in the schemes, preparations and examples below. Those of ordinary skill in the art recognize that the specific synthetic steps of each of the pathways described may be combined in different ways, or with steps from different schemes, to prepare the compounds of the invention or salts thereof. The products of the following steps may be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents are as previously defined unless otherwise indicated. Reagents and starting materials are readily available to those of ordinary skill in the art. Without limiting the scope of the invention, the following schemes, preparations and examples are provided to further illustrate the invention. In addition, it will be appreciated by those of ordinary skill in the art that compounds of formulas XXa, XXb, XXc and XXd can be prepared by using starting materials having the corresponding stereochemical configurations which can be prepared by those of ordinary skill in the art. For example, the following preparation examples use starting materials having a configuration that ultimately corresponds to formula XXa.

Preparation example 12:1- (2-chloro-5, 7-dihydro-6H-pyrrolo [3,4-b ] pyridin-6-yl) ethan-1-one

N, N-diisopropylethylamine (DIPEA, 3.6mL,21 mmol) and acetyl chloride (0.4 mL,6 mmol) were added dropwise to a 0℃solution of 2-chloro-6, 7-dihydro-5H-pyrrolo [3,4-b ] pyridine hydrochloride (1.0 g,5.2 mmol) in dichloromethane (DCM, 13 mL). The reaction mixture was stirred at room temperature for 24 hours. The resulting mixture was diluted with DCM (20 mL) and saturated aqueous NaHCO ₃ (30 mL). The aqueous layer was extracted with DCM (2X 30 mL). The combined organic extracts were dried over MgSO ₄, filtered and concentrated under reduced pressure. The resulting residue was dissolved in DCM, adsorbed onto celite, purified by flash chromatography on silica gel eluting with a gradient of 50-100% acetone in hexane to give the title compound after evaporation of the solvent of the desired chromatographic fraction (0.95 g, 92% yield). ES/MS m/z:197 (M+H).

Preparation example 13: (2S, 4R) -4- ((6-acetyl-6, 7-dihydro-5H-pyrrolo [3,4-b ] pyridin-2-yl) oxy) -2-methylpyrrolidine-1-carboxylic acid tert-butyl ester.

To a solution of (2 s,4 r) -4-hydroxy-2-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester (0.41 g,2.03 mmol), 1- (2-chloro-5, 7-dihydro-6H-pyrrolo [3,4-b ] pyridin-6-yl) ethan-1-one (0.47 g,2.36 mmol) and tetrahydrofuran (THF, 8 mL) was added portionwise potassium tert-butoxide (KO-t-Bu, 0.45g,4 mmol) at Room Temperature (RT) and the mixture stirred at 50 ℃ for 4.5H. The reaction mixture was diluted with water (50 mL) and ethyl acetate (EtOAc, 50 mL). The aqueous layer was extracted with EtOAc (2X 50 mL) and the combined organic extracts were dried over MgSO ₄, filtered and concentrated under reduced pressure. The resulting residue was dissolved in DCM and purified by flash chromatography on silica gel eluting with a gradient of 40-100% acetone in hexane to give the title compound after evaporation of the solvent of the desired chromatographic fraction (0.34 g,47% yield). ES/MS m/z:262 (M+HC ₄H₉).

Preparation example 14:1- (2- (((3 r,5 s) -5-methylpyrrolidin-3-yl) oxy) -5, 7-dihydro-6H-pyrrolo [3,4-b ] pyridin-6-yl) ethan-1-one hydrochloride.

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To a solution of (2 s,4 r) -4- ((6-acetyl-6, 7-dihydro-5H-pyrrolo [3,4-b ] pyridin-2-yl) oxy) -2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (0.34 g,0.94 mmol) in DCM (5.0 mL) was added a solution of 4MHCl in 1, 4-dioxane (1.2 mL,4.8 mmol). The resulting mixture was stirred at room temperature for 3 hours. The resulting suspension was concentrated under reduced pressure and the resulting residue was placed under vacuum for 1 hour to give the title compound (0.28 g, >99% yield). ES/MS m/z:262 (M+H).

Preparation example 15: n- (5-bromo-2, 4-difluoro-phenyl) acetamide.

Acetic anhydride (Ac ₂ O,389 mL) was added to the flask while stirring in a heated block at about 61 ℃ (internal temperature 60 ℃). 5-bromo-2, 4-difluoroaniline (77.7 g,374 mmol) was added in portions to the flask over 30 minutes, maintaining the internal temperature below 65 ℃ during the addition. The reaction mixture was stirred in a heated block at about 61 ℃ for 10 minutes and cooled to room temperature to give a residue which was concentrated from toluene (4 x 200 mL) to give a light brown/pink solid. The concentrated solid was suspended in heptane (80 mL) and the mixture was stirred on a rotary evaporator at 50 ℃ water bath at atmospheric pressure for 15 minutes, cooled to RT, and filtered. The filtered solid was collected and dried in vacuo at 40 ℃ for 2 hours to give the title compound (89.6 g,95% yield) as an off-white solid. ES/MS m/z:250 (M+H).

Preparation example 16: n- (5-bromo-2, 4-difluoro-phenyl) thioacetamide.

To a solution of N- (5-bromo-2, 4-difluoro-phenyl) acetamide (89.6 g, 178 mmol) in anhydrous acetonitrile (ACN, 896 mL) was added pyridin-1-onium-1-yl- [ pyridin-1-onium-1-yl (thio) thiophosphonyl ] sulfanyl-thio-phosphine (68.2 g, 178 mmol, J.Org. Chem.76,1546-1553 (2011)) at room temperature. The slurry was stirred in a 85 ℃ heating block overnight (internal temperature 80 ℃), cooled to RT, and poured into a mixture of ice (200 g) and saturated aqueous NaCl (700 mL). The mixture was diluted with EtOAc (900 mL), stirred at room temperature for 10 min, the layers separated, and the aqueous layer was additionally extracted with EtOAc (900 mL). The combined organic extracts were washed with saturated aqueous NaCl (900 mL), dried over Na ₂SO₄ and concentrated under reduced pressure to give the title compound as a dark brown oil, which was dissolved in DMF (953 mL) at RT and used without additional purification.

Preparation example 17: 5-bromo-6-fluoro-2-methyl-1, 3-benzothiazole.

Sodium tert-butoxide (NaO-t-Bu, 42.6g,430 mmol) was added portionwise to a DMF solution of N- (5-bromo-2, 4-difluoro-phenyl) thioacetamide with stirring over 20 minutes while maintaining the internal temperature below 30 ℃. The reaction mixture was stirred at RT for 5min, stirred in a 42 ℃ heating block (internal temperature 40 ℃) overnight, and cooled to RT. The reaction mixture was added dropwise to a mixture of ice (250 g) and H ₂ O (700 mL) over 5 minutes, keeping the internal temperature below 20 ℃. The mixture was stirred for 10 min at RT and filtered. The filtered solid was dried overnight in vacuo at 40℃and suspended in 50% MeOH/H ₂ O (480 mL). The mixture was stirred in a 45 ℃ heating block for 15min, cooled to RT, and filtered. The filtered solid was dried in vacuo at 40 ℃ for 72 hours to give a light brown solid. The material was combined with EtOAc (700 mL) and the mixture stirred at RT for 10 min, H ₂ O (700 mL) was added, and the layers separated. The aqueous layer was extracted with EtOAc (700 mL), and the combined organic extracts were washed with saturated aqueous NaCl (700 mL), dried over MgSO ₄, and concentrated under reduced pressure to give the title compound (62.7 g,71% yield) as a brown solid. ¹H NMR(d₆ DMSO) delta 2.82 (s, 3H), 7.57 (m, 1H), 8.12 (m, 1H). Preparation example 18: 6-fluoro-2-methyl-1, 3-benzothiazole-5-carbaldehyde.

5-Bromo-6-fluoro-2-methyl-1, 3-benzothiazole (100.9 g,410 mmol) in DMF (1009 mL) was bubbled with N ₂ for 5 min at room temperature with stirring. Potassium formate (52.3 g,615.0 mmol), palladium (II) acetate (2.82 g,12.30 mmol), 2- (di-tert-butylphosphino) biphenyl (5.19 g,17.2 mmol) and 1, 3-tetramethylbutyl isocyanide (90.8 mL,492.0 mmol) were added and the mixture was bubbled with N ₂ at room temperature under stirring for 30 min. The reaction mixture was stirred at an internal temperature of 65 ℃ overnight, cooled to 20-25 ℃ and 2M aqueous HCl (820 mL) was added dropwise over 30 minutes keeping the internal temperature below 30 ℃. The resulting mixture was stirred at 20-25℃for 2 hours and diluted with EtOAc (1.5L) and H ₂ O (1L). The layers were separated and the organic layer was washed with 10% aqueous n-acetyl-cysteine (2×1L), saturated aqueous Na ₂CO₃ (750 ml×2) and saturated aqueous NaCl (750 mL); the organic extract was dried over MgSO ₄ and concentrated under reduced pressure to give a first crude material. The HCl aqueous layer from the first extraction was further extracted with EtOAc (1L, then 500 mL) and the combined organic extracts were washed with saturated aqueous NaCl (500 mL), dried over MgSO ₄ and concentrated under reduced pressure to give a second crude material. The combined N-acetyl-cysteine aqueous layer was then extracted with EtOAc (1L, then 500 mL) and the combined organic extracts were washed sequentially with saturated aqueous Na ₂CO₃ (500 mL) and saturated aqueous NaCl (500 mL); the combined organic extracts were dried over MgSO ₄ and concentrated under reduced pressure to give a third crude material. Three batches of crude material were combined in methyl tert-butyl ether (MTBE, 250 mL) and heptane (250 mL) and the resulting slurry was stirred at RT for 20 min. The resulting precipitate was filtered and washed with heptane (250 mL). The filtered solid was dried in vacuo at 45 ℃ to give the first batch product. The filtrate was concentrated and the residue purified by column chromatography on silica eluting with a gradient of 0-100% etoac/heptane. The product containing fractions were combined and concentrated to a volume of about 400mL, the resulting slurry was stirred at RT for 15 min, filtered, and the filtered solid was washed with heptane (200 mL) to give a second batch of product. The first and second batch products were combined with heptane (500 mL), slurried at room temperature, filtered, and the filtered solids washed with heptane (250 mL). The filtered solid was dried under vacuum at 45 ℃ overnight to give the title compound (63.5 g,79% yield). ES/MS M/z 196 (M+H).

It will be appreciated that the individual isomers, enantiomers and diastereomers of the OGA inhibitor compounds provided herein as part of the novel combinations and methods of the invention may be separated or resolved by methods such as selective crystallization techniques or chiral chromatography by one of ordinary skill in the art at any convenient point (see, e.g., j. Jacques, et al ,"Enantiomers,Racemates,and Resolutions,"John Wiley and Sons,Inc.,1981,and E.L.Eliel and S.H.Wilen,"Stereochemistry of Organic Compounds,"Wiley-Interscience,1994).

Pharmaceutically acceptable salts of the OGA inhibitor compounds of the present invention can be formed, for example, by reacting the appropriate free base of the compounds of the present invention with an appropriate pharmaceutically acceptable acid in an appropriate solvent under standard conditions well known in the art. The formation of such salts is well known and understood in the art. See, e.g., ,Gould,P.L.,"Salt selection for basic drugs,"International Journal of Pharmaceutics,33:201-217(1986);Bastin,R.J. et al ,"Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,"Organic Process Research and Development,4:427-435(2000); and Berge, s.m. et al, "Pharmaceutical Salts," Journal of Pharmaceutical Sciences,66:1-19, (1977).

Example 1: synthesis of the OGA inhibitor N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide (formula Ia).

This compound and its synthesis are disclosed in U.S. patent No. US10,081,625, the entire contents of which are incorporated herein by reference.

N- (4-fluoro-5-formyl-thiazol-2-yl) acetamide (28.3 g,150 mmol) was added to 5-methyl-3- [ [ (2S, 4S) -2-methyl-4-piperidinyl ] oxymethyl ] -1,2, 4-oxadiazole hydrochloride (48.7 g,185mmol,94% purity) in ethyl acetate (707 mL) at room temperature. The reaction mixture was stirred at room temperature and N, N-diisopropylethylamine (34.1 mL,195 mmol) was added dropwise over 1 minute, and sodium triacetoxyborohydride (98.5 g, 457mmol) was added in one portion. The reaction mixture was stirred overnight in a 31 ℃ heating block at an internal temperature of 30 ℃ and cooled to an internal temperature of 5 ℃ in an ice water bath. To the mixture was added 2M aqueous hydrochloric acid (226 mL) over 15 minutes, keeping the internal temperature below 10 ℃. Water (250 mL) was added to the mixture and the mixture was stirred at room temperature for 5 min. The layers were separated and the organic layer was extracted with a mixture of 2M aqueous hydrochloric acid (28 mL) in water (50 mL). The first aqueous layer was stirred in an ice-water bath and 50% aqueous sodium hydroxide (25.7 mL) was added dropwise over 10 minutes maintaining the internal temperature below 10 ℃. The mixture was diluted with saturated aqueous sodium bicarbonate (100 mL), stirred at room temperature for 10 min, and extracted with ethyl acetate (3×400 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated to give a residue. The second aqueous layer, extracted with aqueous hydrochloric acid, was diluted with 2-methyltetrahydrofuran (200 mL) and the mixture was passed through a short pad of celite. The filtrate was transferred to a separatory funnel and the layers were separated. The aqueous layer was stirred in an ice water bath and 50% aqueous sodium hydroxide solution (3.15 mL) was added dropwise over 5 minutes maintaining the internal temperature below 10 ℃. The mixture was diluted with saturated aqueous sodium bicarbonate (10 mL), stirred at room temperature for 5 min, and extracted sequentially with ethyl acetate (3×40 mL) and 10% isopropanol in ethyl acetate (100 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated to give a residue which was combined with the residue from the first part of the work-up. The combined residue was passed through a pad of silica gel (350 g), eluted with ethyl acetate (3.5L), and the filtrate was concentrated to give a residue (45.8 g). The residue (47.5 g combined batch, 123.9 mmol) was purified by flash chromatography on silica gel eluting with 50-100% ethyl acetate in heptane. The product containing fractions were concentrated to give a residue, which was suspended in a 1:1 mixture of methyl tert-butyl ether and heptane (448 mL). The mixture was stirred in a 46 ℃ heating block at an internal temperature of 45 ℃ for 30 minutes and cooled to room temperature over 2 hours with stirring. The mixture was filtered and the collected solids were washed with a 1:1 mixture of methyl t-butyl ether and heptane (30 mL). The filtered solid was dried under vacuum at 40 ℃ overnight to give the title compound (28.5 g,49% yield). MS M/z 384.0 (M+H). Optical rotation: [ α ] _D ²⁰ = +33.4 ° (c=0.26, methanol).

Alternative synthesis of N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide.

To a solution of N- (4-fluoro-5-formyl-thiazol-2-yl) acetamide (50 mg,0.28 mmol) and 5-methyl-3- [ [ (2S, 4S) -2-methyl-4- [ piperidinyl ] oxymethyl ] -1,2, 4-oxadiazole (40 mg,0.19 mmol) in dichloromethane (10 mL) under nitrogen was added N, N-diisopropylethylamine (0.1 mL,0.57 mmol) and sodium triacetoxyborohydride (120 mg,0.57 mmol). The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was poured into saturated aqueous sodium bicarbonate (10 mL). The layers were separated and the aqueous phase extracted with dichloromethane (2X 10 mL). The combined organic extracts were dried over magnesium sulfate, filtered and concentrated under reduced pressure to give an orange oil. The residue was dissolved in methanol (to a total volume of 9.8 mL), filtered, and purified by preparative HPLC (Phenomenex Gemini-NX 10Micron 50x 150mm C-18) eluting with a gradient of 15% (CH ₃ CN & water (containing 10mM ammonium bicarbonate), adjusted to pH9 with ammonium hydroxide) to 100% CH ₃ CN for 10 minutes (110 mL/min,1 sample introduction, 271/204 nm) to give the title compound (20 mg,28% yield). MS M/z 384.2 (M+H).

Example 1A: crystalline N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide.

Crude N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide (29.9 g) was suspended in 447 mL of 50% methyl tert-butyl ether in heptane for 30min at 46 ℃. The mixture was stirred and cooled to 19 ℃ over two hours, then filtered, followed by washing with 30ml of 50% methyl tert-butyl ether in heptane to give the title compound (28.5 g,95% yield).

X-ray powder diffraction (XRPD) of example 1A.

The XRPD pattern of the crystalline solid was that provided with a CuKa sourceAnd Vantec detector, bruker D4 Endeanor X-ray powder diffractometer operating at 35kV and 50 mA. The sample was scanned at a step size of 0.0087 ° for 2θ,0.5 seconds/step for scan rate, 0.6mm for divergence angle, 5.28mm for fixed anti-scatter length, and 9.5mm for detector slit at 2θ between 4 and 40 °. The dry powder was mounted on a quartz sample holder and a slide was used to obtain a smooth surface. As is well known in the crystallography arts, for any given crystal form, the relative intensities of diffraction peaks may vary due to the preferred orientation resulting from factors such as crystal morphology and habit. When the preferential orientation effect is present, the peak intensity changes, but the characteristic peak position of the polymorph is unchanged. (see, e.g., ,The U.S.Pharmacopeia 38-National Formulary 35Chapter 941Characterization of crystalline and partially crystalline solids by X-ray powder diffraction(XRPD)Official May 1,2015).. Furthermore, the field of crystallography also knows that the angular peak position may vary slightly for any given crystal form. For example, the peak position may vary due to variations in temperature or humidity of the analytical sample, sample displacement, or the presence or absence of internal standards. In this example, variations in peak position of + -0.2 in 2. Theta. Will take into account these potential variations without impeding the clear identification of the illustrated crystal form. Confirmation of the crystal form may be based on distinguishing any unique combination of peaks (in degrees 2. Theta.) typically more prominent peaks. Diffraction patterns of the crystal form collected at ambient temperature and relative humidity are adjusted based on NIST 675 standard peaks at 8.85 and 26.77 degrees 2. Theta. In this example, the present invention is not limited to the use of such a system.

The crystalline N- [ 4-fluoro-5- [ [ (2 s,4 s) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide samples prepared were characterized by XRPD patterns using CuKa radiation, with diffraction peaks (2-theta values) as described in table 1 below. Specifically, the profile comprises a combination of a peak of 12.1 ° and one or more peaks selected from 15.3 °, 21.6 °, 22.2 °, 22.7 °, 23.5 °, 24.3 ° and 26.8 °, and the diffraction angle error is 0.2 °.

Table 1: x-ray powder diffraction peak of crystalline N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide of example 1A.

Example 2: synthesis of the OGA inhibitor N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide (formula Xa).

To a solution of 2-methyl-5- [ [ (2S, 4S) -2-methyl-4-piperidinyl ] oxymethyl ] -1,3, 4-oxadiazole 2, 2-trifluoro acetic acid (160 mg,0.7 mmol) in ethyl acetate (1 mL) under nitrogen was added N, N-diisopropylethylamine (210 mL,0.12 mmol) and the solution was stirred for 5 min. N- (4-fluoro-5-formyl-thiazol-2-yl) acetamide (40 mg,0.12 mmol) was added and stirred for 5 min, sodium triacetoxyborohydride (55 mg,0.25 mmol) was added and the reaction mixture was heated to 40℃and stirred overnight. The mixture was concentrated under reduced pressure to give a brown solid. The residue was dissolved in dimethyl sulfoxide (to a total volume of 1 mL) and purified by preparative HPLC (Phenomenex Gemini-NX 10Micron 30x 100mm C-18) eluting with a gradient of 15% (CH ₃ CN & water (containing 10mM ammonium bicarbonate), adjusted to pH9 with ammonium hydroxide) to 100% CH ₃ CN for 12 min (100 mL/min,1 sample introduction, 271/204 nm) to give the title compound (7 mg,14% yield). MS M/z 384.2 (M+H).

Alternative synthesis of crystalline N- [ 4-fluoro-5- [ [ (2 s,4 s) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide.

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Sodium triacetoxyborohydride (59.1 g,279 mmol) was added to a mixture of 2-methyl-5- [ [ (2S, 4S) -2-methyl-4-piperidinyl ] oxymethyl ] -1,3, 4-oxadiazole (23.3 g,93.0 mmol), ethyl acetate (438 mL) and N, N-diisopropylethylamine (32.4 mL,186 mmol) at room temperature. The reaction mixture was stirred in a 31℃heating block for 15 minutes at an internal temperature of 30℃and then N- (4-fluoro-5-formyl-thiazol-2-yl) acetamide (17.5 g,93.0 mmol) was added in portions over 5 minutes. The reaction mixture was stirred overnight in a 31 ℃ heating block at an internal temperature of 30 ℃ and cooled to an internal temperature of 5 ℃ in an ice water bath. To the mixture was added 2M aqueous hydrochloric acid (140 mL) over 15 minutes, keeping the internal temperature below 10 ℃. The mixture was stirred at room temperature for 15 min, diluted with water (50 mL) and ethyl acetate (20 mL), and the layers were separated. The organic layer was extracted with a mixture of 2M aqueous hydrochloric acid (35 mL) in water (100 mL). The combined aqueous layers were stirred in an ice-water bath and 50% aqueous sodium hydroxide (19.5 mL) was added dropwise over 10 minutes maintaining the internal temperature below 10 ℃. The mixture was diluted with saturated aqueous sodium bicarbonate (50 mL) and extracted with 2-methyltetrahydrofuran (3×200 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated to give a residue which was purified by flash chromatography on silica gel eluting with 0-15% 2-propanol in dichloromethane. The product containing fractions were concentrated to give a residue which was concentrated from heptane (100 mL). The concentrated material was combined with 40% ethyl acetate in heptane (457 mL) and the mixture stirred in a 50 ℃ heating block for 1 hour, cooled to room temperature, and filtered. The filtered solid was dried in vacuo at 40℃for 1 hour to give a first crop (22.9 g). The filtrate was concentrated to give a residue which was combined with 40% ethyl acetate in heptane (50 mL) and the mixture was stirred in a 50 ℃ heating block for 30 min, cooled to room temperature and filtered. The filtered solid was combined with 50% ethyl acetate in heptane (33 mL) and the mixture stirred in a 50 ℃ heating block for 1 hour, cooled to room temperature and filtered. The filtered solid was dried in vacuo at 40℃for 1 hour to give a second crop (2.50 g).

A batch combination comprising the first and second batch products (29.3 g) was combined with ethyl acetate (117 mL) and heptane (117 mL) at room temperature. The mixture was stirred in a 51 ℃ heating block for 30 minutes at an internal temperature of 50 ℃, then cooled to room temperature and filtered. The filtered solid was dried under vacuum at 40 ℃ overnight to give the title compound (26.7 g,75% yield) as a crystalline solid. MS M/z 384.0 (M+H). Optical rotation: [ α ] _D ²⁰ = +39° (c=0.2, methanol).

X-ray powder diffraction (XRPD) of crystalline N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide.

Crystalline N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide (218 mg) was dissolved in 1.25mL of methanol at 60℃for 5 minutes. The solution was cooled to ambient temperature and stirred for 20 minutes. The resulting solid was isolated by vacuum filtration. The final solid product was 163mg or 75% yield.

The XRPD pattern of the crystalline solid was that provided with a CuKa sourceAnd Vantec detector, bruker D4 Endeanor X-ray powder diffractometer operating at 35kV and 50 mA. The sample was scanned at a step size of 0.0087 ° for 2θ,0.5 seconds/step for scan rate, 0.6mm for divergence angle, 5.28mm for fixed anti-scatter length, and 9.5mm for detector slit at 2θ between 4 and 40 °. The dry powder was mounted on a quartz sample holder and a slide was used to obtain a smooth surface. As is well known in the crystallography arts, for any given crystal form, the relative intensities of diffraction peaks may vary due to the preferred orientation resulting from factors such as crystal morphology and habit. When the preferential orientation effect is present, the peak intensity changes, but the characteristic peak position of the polymorph is unchanged. See, e.g., ,The U.S.Pharmacopeia 38-National Formulary 35Chapter<941>Characterization of crystalline and partially crystalline solids by X-ray powder diffraction(XRPD)Official May 1,2015., furthermore, the field of crystallography, as well known, the angular peak position may vary slightly for any given crystal form. For example, the peak position may change due to a change in temperature or humidity of the analysis sample, sample displacement, or the presence or absence of an internal standard. In this example, peak position variations of + -0.2 in 2 theta will take these potential variations into account without impeding the clear identification of the crystal form shown. The validation of the crystalline form may be based on distinguishing any unique combination of peaks (in degrees 2 theta), typically the more prominent peaks. The diffraction patterns of the crystalline forms collected at ambient temperature and relative humidity were adjusted based on the NIST 675 standard peaks at 8.85 and 26.77 degrees 2θ.

Thus, crystalline N- [ 4-fluoro-5- [ [ (2 s,4 s) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide was characterized by XRPD pattern using CuKa radiation, having diffraction peaks (2-theta values) as described in table 2. More specifically, the profile preferably comprises a combination of a peak at 13.5 ° with one or more peaks selected from 5.8 °, 13.0 °, 14.3 °, 17.5 °, 20.4 °, 21.4 ° and 22.2 °, with a diffraction angle error of 0.2 °.

Table 2: x-ray powder diffraction peak of crystalline N- [ 4-fluoro-5- [ [ (2 s,4 s) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide.

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In vitro human OGA enzyme assay

OGA protein production

The nucleotide sequence encoding full length human O-GlcNAc- β -N-acetylglucosaminidase (NM-012315) was inserted into the pFastBac1 (Invitrogen) vector with an N-terminal polyhistidine (HIS) tag. Baculovirus production was performed according to the Bac-to-Bac baculovirus expression system (Invitrogen) protocol. Sf9 cells were infected with 10mL P1 virus per liter of culture at 1.5×10 ⁶ cells/mL and incubated at 28 ℃ for 48 hours. The cells were centrifuged, washed with PBS, and the pellet was stored at-80 ℃.

Purification of the above OGA protein (His-OGA) was as follows: 4L of cells were lysed in 200mL of buffer containing 50mM Tris, pH 8.0, 300mM NaCl, 10% glycerol, 10mM imidazole, 1mM Dithiothreitol (DTT), 0.1% Triton ^TM X-100, 4 pieces of protease inhibitor (complete free of EDTA, roche) for 45 min at 4 ℃. The cell lysate was then spun at 16500rpm for 40 minutes at 4℃and the supernatant incubated with 6mL of Ni-NTA resin (nickel-nitrilotriacetic acid) for 2 hours at 4 ℃.

The resin was then packed onto the column and washed with 50mM Tris, pH8.0, 300mM NaCl, 10% glycerol, 10mM imidazole, 0.1% Triton ^TM X-100, 1mM DTT followed by 50mM Tris, pH8.0, 150mM NaCl, 10mM imidazole, 10% glycerol, 1mM DTT. Proteins were eluted with 50mM Tris, pH8.0, 150mM NaCl, 300mM imidazole, 10% glycerol, 1mM DTT. The combined His-OGA-containing fractions were concentrated to 6mL and loaded onto Superdex75 (16/60). Proteins were eluted with 50mM Tris, pH8.0, 150mM NaCl, 10% glycerol, 2mM DTT. His-OGA-containing fractions were pooled and protein concentration was measured using BCA (Bradford colorimetric assay).

OGA enzyme assay

The OGA enzyme catalyzes the removal of O-GlcNAc from nuclear cytoplasmic proteins. To measure this activity, fluorescein di-N-acetyl- β -N-acetyl-D-aminoglycoside (FD-GlcNAc, kim et al Carbohydrate Research (2006), 341 (8), 971-982) was used as substrate at a final concentration of 10. Mu.M (96 well assay format) or 6.7. Mu.M (384 well assay format). This fluorogenic substrate fluoresces after cleavage by OGA and thus enzyme activity can be measured by the increase in fluorescence detected at 535nm (excitation at 485 nm).

Assay buffer was formulated to give a final concentration of 50mM H ₂NaPO₃-HNa₂PO₃, 0.01% bovine serum albumin and 0.01% Triton ^TM X-100 in water at pH 7. The final enzyme concentration was 3nM (in the 96-well assay format) or 3.24nM (in the 384-well assay format). Both assay formats produced substantially identical results.

Test compounds were diluted in pure dimethyl sulfoxide (DMSO) using a ten-point concentration response curve. The maximum compound concentration in the reaction mixture was 30. Mu.M. The appropriate concentration of compound was pre-incubated with the OGA enzyme for 30 minutes, and then the reaction was started by adding substrate. The reaction was carried out at room temperature for 60 minutes. The fluorescence was then read without stopping the reaction. IC ₅₀ values were calculated by plotting the normalized data vs. log of the compound and fitting the data using a four parameter logistic equation.

The compound of example 1 was tested essentially as described above and exhibited an IC ₅₀ of 1.97nm±1.22 (n=9). This data demonstrates that the compound of example 1 inhibits OGA enzyme activity in vitro.

The compound of example 2 was also tested essentially as described above and exhibited an IC ₅₀ of 2.13nm±0.89 (n=5). This result demonstrates that the compound of example 2 inhibits OGA enzyme activity in vitro.

Whole cell assay for measuring OGA enzyme activity inhibition

Cell plating:

TRex-293 cells modified to induce expression of the P301S-1N4R form of microtubule-associated protein tau were generated and maintained in growth medium consisting of DMEM high glucose (Sigma #D5796), supplemented with 10% tetracycline free fetal bovine serum (FBS, sigma F2442), 20mM HEPES, 5 μg/mL blasticidin (Life Technologies #A11139-03) and 200 μg/mL Zeocin (Life Technologies #R250-01) using standard conditions known in the art. In the experiments, cells were plated at a density of 10,000-14,000 cells per well in Corning Biocoat (356663) 384-well plates coated with poly-D-lysine and incubated in a cell incubator at 37 ℃/5% co ₂ for 20-24 hours. The experiment was performed without inducing Tau expression.

Compound treatment:

Test compounds were serially diluted 1/3 in pure DMSO using a ten-point concentration response curve and further diluted in growth medium. 20-24 hours after plating, cells are treated with test compounds in growth medium; the maximum compound concentration was 15 μm (0.15% dmso). Maximum inhibition was defined by repeated measures of 15 μ M THIAMET G and minimum inhibition was defined by repeated measures of 0.15% dmso treatment. The cells were returned to the incubator at 37℃C.5% CO ₂ for 20-24 hours. Compounds were tested in duplicate in each plate.

Immunostaining:

After 20-24 hours of compound treatment, the medium was removed from the assay plate and 25 μl of 3.7% formaldehyde solution (Sigma #f1635) in DPBS (Sigma #d8537; dulbecco phosphate buffered saline) was added to each well and incubated for 30 minutes. Cells were then washed once with DPBS and permeabilized with 0.1% Triton ^TM X-100 (Sigma #T9284). After 30min, cells were washed twice with DPBS, and blocking solution (1% BSA/DPBS/0.1% Triton ^TM X-100) was added to each well and incubated for 60 min. The blocking solution was removed and a solution of 0.40-0.33. Mu.g/mL O-GlcNAc protein antibody (RL 2 clone, thermo, MA 1072) in the blocking solution was added to the cells and allowed to stand overnight at 2-8 ℃. The next day, cells were washed twice with DPBS, and 2 μg/mL of secondary antibody Alexa Fluor 488 goat anti-mouse IgG (Life Technologies # a 11001) in DPBS was added to each well and allowed to stand at room temperature for 90 minutes. The secondary antibody was removed, the cells were washed twice with DPBS, and DAPI (Sigma #D9564;4', 6-diamidino-2-phenylindole, dilactate) and RNase (Sigma, R6513) in solution in DPBS at concentrations of 1 and 50. Mu.g/mL were added to each well, respectively. Plates were sealed, incubated for one hour and analyzed on Acumen eX3 hci (TTP Labtech). All incubation and washing steps described above were performed at room temperature, except for the primary antibody.

Analysis and results:

Plates were analyzed on an Acumen eX3 instrument using 488 and 405nm excitation lasers and two emission filters FL2 (500-530 nm) and FL1 (420-490 nm). The FL2 filter is the signal corresponding to the O-GlcNAc protein antibody (RL 2 clone) and the FL1 filter is the signal corresponding to the nucleus (DAPI). The ratio of total FL 2/total FL1 (total fluorescence per well without subject or population selection) was used for data analysis. Data were normalized to the maximum inhibition referenced by 15 μ M THIMAMET G treatment and the minimum inhibition obtained by 0.15% dmso treatment. The data was fitted using a nonlinear curve fitting application (4 parameter logistic equation) and IC ₅₀ values were calculated and reported.

The compound of example 1 was tested essentially as described above and exhibited an IC ₅₀ of 21.9nm±7.3 (n=5). This data demonstrates that the compound of example 1 inhibits OGA enzyme activity in a cellular assay.

The compound of example 2 was also tested essentially as described above and exhibited an IC ₅₀ of 22.6nm±7.3 (n=3). This result demonstrates that the compound of example 2 inhibits OGA enzyme activity in a cellular assay.

Single dose escalation studies in healthy individuals to assess the safety and pharmacokinetics of the compound of example 1

Phase 1, single-center, individual and investigator blinded, single escalated dose, placebo-controlled, crossover, randomized study was performed to evaluate the safety, tolerability and Pharmacokinetics (PK) of the compound of example 1 in healthy individuals. The study was performed in 2 alternating groups (groups 1 and 2), up to 3 study periods, involving 6 dose levels. Individuals were randomly assigned to 1 of 3 treatment sequences in each group, each sequence comprising 2 doses of the compound of example 1 and 1 placebo dose in a completely crossed fashion over 3 study periods. The clinical study design is summarized in table 3.

Table 3. Clinical study design.

	Group 1	Group 2
			Stage 1	0.15Mg of the compound of example 1 or placebo	---
Stage 1	Cleaning for 7 days or more	0.6Mg of the compound of example 1 or placebo
			Stage 2	2Mg of the compound of example 1 or placebo	Cleaning for 7 days or more
Stage 2	Cleaning for 7 days or more	5Mg of the compound of example 1 or placebo
			Stage 3	10Mg of the compound of example 1 or placebo	Cleaning for 7 days or more
Stage 3	---	16Mg of the compound of example 1 or placebo

After a fast of at least 8 hours overnight, the oral capsule was administered in the morning of each dosing day with about 240mL of room temperature water taken in a sitting position. Similar to oral capsule administration, a dose of 0.15mg to 2mg was administered as an oral solution of the compound of example 1 with water via an oral administration syringe. Tables 4a and 4b summarize the treatment regimens.

Table 4a. Treatment regimen for the compound of example 1.

Dose intensity (mg)	0.15 To 2	5 To 16
			Dosage formulation	Oral solution	Capsule
Dosage of	Temporary formulations	Temporary formulations
			Route of administration	Oral administration	Oral administration
Dose description	Single dose	Single dose

Table 4b. Placebo treatment regimen.

Dose intensity (mg)	Is not suitable for	Is not suitable for
			Dosage formulation	Oral liquid (Carrier)	Capsule (HPMC ¹)
Dosage of	Matched placebo	Matched placebo
			Route of administration	Oral administration	Oral administration
Dose description	Single dose	Single dose

¹ HPMC = hypromellose

Capsules containing the compound of example 1 were prepared extemporaneously. An oral dose of 0.15mg to 2mg of the compound of example 1 was temporarily prepared as a drug in solution. The total number of capsules administered for any particular group/dosing period was the same for all individuals, whether assigned to placebo or the compound of example 1. However, the number of capsules may vary between dosing periods and groups. This is similar to the case where an orally administered solution is prepared temporarily to maintain the blindness. The compound of example 1 was provided as the free base, free of inactive ingredients for temporary preparation. A matched volume of oral solution vehicle without compound of example 1 was used as placebo at a dose of 0.15mg to 2 mg.

An approximately 3mL venous blood sample was collected to determine the plasma concentration of the compound of example 1. The concentration of the compound of example 1 was determined using validated liquid chromatography and tandem mass spectrometry methods. PK parameter estimates for the compound of example 1 were calculated using standard non-compartmental analysis methods. The plasma concentrations of the compound of example 1 were summarized by the dose of the compound of example 1 administered and the approximate time of PK blood sample collection. The PK data for a single incremental dose of the compound of example 1 in healthy individuals, substantially as described above, are presented in tables 5 and 6.

Table 5 mean plasma concentrations for single escalation dose studies following oral administration of the compound of example 1 in healthy individuals.

Table 6. PK parameters ¹ for the compound of example 1 following oral administration in healthy individuals.

¹ Unless otherwise indicated, data are expressed as geometric mean (%CV geometric mean)

² Application amount of the Compound of example 1

³ N = number of individuals

⁴ CL/F = apparent clearance calculated after oral administration

⁵ AUC (0- ≡) =area under concentration versus time curve from time zero to infinity

⁶ Cmax = maximum drug concentration observed

⁷V_z Apparent distribution volume after oral administration/F =

⁸t_1/2 Terminal half-life (geometric mean (min-max))

⁹t_max Time of maximum drug concentration observed; median (min-max)

¹⁰ Amount of compound of example 1 excreted in urine up to 24 hours after administration of Ae0-24 =

¹¹ CLr = renal clearance rate

¹²N＝5

¹³N＝3

¹⁴ Nc=not calculated

The data discloses that AUC _(0-∞) and C _max of the compound of example 1 increased approximately in proportion to the dose over the 0.6mg to 16mg dose range. For the compound of example 1, median t _max is about 1 hour and t _1/2 is about 6 hours.

Evaluation of brain O-GlcNAc enzyme occupancy after a single oral dose of the compound of example 1 by measuring radioligand [ ¹⁸ F ] LSN3316612 for positron emission tomography in healthy individuals

Brain penetration and target engagement of brain O-GLCNACASE (OGA) after a single oral dose of 0.25mg, 1mg, and 5mg of N- [ 4-fluoro-5- [ [ (2S, 4S) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide (a compound of formula I) was demonstrated by one of ordinary skill in the art essentially as described below for single-site, open-label, non-random, positron Emission Tomography (PET) studies. [ ¹⁸ F ] LSN3316612 is a positron-emitting radiopharmaceutical for in vivo imaging of OGA in the brain and is used to evaluate target engagement of compounds that inhibit OGA. ¹⁸ The preparation and use of F-LSN3316612 as a PET radioligand is known in the art, for example as described in S.Lu et al, science Translational Medicine,12, (2020). This single dose PET study using [1 ^8F ] LSN3316612 tracer evaluates brain OGA Enzyme Occupancy (EO) over a suitable range of doses that have proven to be safe and well tolerated.

Healthy individuals were assigned to 1 out of 4 groups, with 4 individuals in each group completing the study. All individuals received one baseline PET scan and two post-dosing PET scans. A baseline PET scan was performed up to about 14 days prior to administration of the compound of example 1. In summary, each individual received a single dose of the compound of example 1 and 3 administrations of [ ¹⁸ F ] LSN3316612 PET tracer. Administration of the compound of example 1 was performed after the baseline PET scan was completed. For the 0.25mg and 5mg doses of the compound of example 1, scans were performed at about 2 hours and 24 hours after administration, and for the 1mg dose of the compound of example 1, scans were performed at about 2 hours and 24 hours or 30 hours and 54 hours after administration. Dynamic PET data of the brain were acquired within 120min immediately after tracer injection. EO is summarized by the compound dose and approximate scan time of example 1.

For doses of ≡ 3mg, the compound of example 1 was orally administered in the form of a capsule preparation. For doses below 3mg, the compound of example 1 is weighed into a suitable container and dissolved in a suitable volume of deaerationOr in a diluent.

[ ¹⁸ F ] LSN3316612 was generated from non-radioactive precursors in clinical field radiochemical facilities on the day of each PET scan. [ ¹⁸ F ] LSN3316612 injection is a clear solution for intravenous injection, which is prepared from physiological saline containing ethanol, sterile water for injection and sodium ascorbate. [ ¹⁸ F ] LSN3316612 was delivered as formulated physiological saline (0.9% NaCl) with the objective of containing about 3.3% (v/v) ethanol (EtOH) and sodium ascorbate (4.67 mg/mL). The [ ¹⁸ F ] LSN3316612 was administered intravenously during a 3 minute infusion using an infusion pump and then rinsed with 10mL saline. Prior to PET imaging, an individual is catheterized (for radiotracer infusion) according to standard clinical practice. Each individual received one [ ¹⁸ F ] LSN3316612 injection at each imaging visit. The dose of the intravenous radiopharmaceutical is about 5mCi (no more than 6 mCi), the maximum mass dose is 10 μg, and the maximum volume is 10mL.

For each PET scan, the radiochemistry laboratory synthesizes radioligands from the precursors according to PET unit production protocols known in the art, for example as described in Lee, j, liow, j, paul, s. Et al ,"PET Quantification of Brain O-GlcNAcase With[¹⁸F]LSN3316612in Healthy Human Volunteers,"EJNMMI Res 10,20(2020).

Arterial blood samples were collected from all individuals during each PET scan to measure radioactivity and provide input for PET tracer kinetic analysis. Venous blood samples were collected after administration of the compound of example 1 to measure plasma concentrations of the compound of example 1 using validated liquid chromatography and tandem mass spectrometry analysis.

The primary imaging result of [ ¹⁸ F ] LSN3316612 PET is the total distributed volume (V _T) determined in the areas where OGA is present, including cortical areas, basal ganglion areas, thalamus and cerebellum. Temporal activity data using attenuation correction in different brain regions is analyzed. The imaging data was analyzed with a 2-tissue compartment model with arterial input function to determine V _T. The OGA EO after a single dose of the compound of example 1 was obtained using a graphical analysis from the occupancy map:

V _T (baseline) -V _T (dosing) =occupancy x (V _T (baseline) -V _ND),

Wherein V _T (baseline) and V _T (dosing) are total distribution volumes in several areas obtained after baseline and compound of example 1 administration, respectively. Occupancy is determined as the slope of the linear regression of the curve, while the indivisible distribution volume V _ND is taken as the x-intercept.

Target engagement of brain OGA following a single oral dose of 0.25mg, 1mg and 5mg of the compound of example 1, substantially as described above, is shown in tables 7a-7 c.

Table 7a: brain OGA occupancy of the compound of example 1 at 0.25mg dose.

Table 7b: brain OGA occupancy of the compound of example 1 at 1mg dose.

Table 7c: brain OGA occupancy of the compound of example 1 at 5mg dose.

The data provided in tables 7a-7c disclose a plasma concentration dependent change in brain OGA EO, wherein the 5mg dose of the compound of example 1 has EO over 90% EO 24 hours after administration. It was found that the compound of example 1 at a dose of 1mg was 80.6% EO 24 hours after administration and 30.3% EO 54 hours after administration. The compound of example 1 was found to be 46% eo 24 hours after administration at a dose of 0.25 mg.

PK data from a single escalation dose study of the compound of example 1 in healthy individuals and PET studies demonstrating brain penetration and target engagement of brain OGA after a single oral dose of the compound of example 1 as described above, low doses and dose regimens for treating neurodegenerative diseases including AD and other neurodegenerative tauopathies with the compound of example 1 are listed below:

the total dose of the compound of example 1 was 0.25 mg/day to 5 mg/day.

The total dose of the compound of example 1 was 0.1 mg/day to 3 mg/day.

The total dose of the compound of example 1 was 0.25 mg/day to 3 mg/day.

The total dose of the compound of example 1 was 0.1 mg/day to 2 mg/day.

The total dose of the compound of example 1 was 0.25 mg/day to 2 mg/day.

The total dose of the compound of example 1 was 0.1 mg/day to 1 mg/day.

The total dose of the compound of example 1 was 0.25 mg/day to 1 mg/day.

The total dose of the compound of example 1 was 5 mg/day.

The total dose of the compound of example 1 was 3 mg/day.

The total dose of the compound of example 1 was 2 mg/day.

The total dose of the compound of example 1 was 1 mg/day.

The total dose of the compound of example 1 was 0.25 mg/day.

The total dose of the compound of example 1 was 0.1 mg/day.

The total daily dose of the compound of example 1 is preferably one unit dose.

It is further preferred that the total daily dose of the compound of example 1 administered is two unit doses. Preferably, the total daily dose of the compound of example 1 is administered in two unit doses, wherein each dose contains an equal amount of the compound of example 1. Preferably, when the total daily dose of the compound of example 1 is administered in two unit doses, the administration of each unit dose is at least 8 hours apart.

In addition, the total dose of the compound of example 1 in the pharmaceutical composition is as follows:

The total dose of the compound of example 1 was 0.25mg to 5mg.

The total dose of the compound of example 1 was 0.1mg to 3mg.

The total dose of the compound of example 1 was 0.25mg to 3mg.

The total dose of the compound of example 1 was 0.1mg to 2mg.

The total dose of the compound of example 1 was 0.25mg to 2mg.

The total dose of the compound of example 1 was 0.1mg to 1mg.

The total dose of the compound of example 1 was 0.25mg to 1mg.

The total dose of the compound of example 1 was 3mg.

The total dose of the compound of example 1 was 2mg.

The total dose of the compound of example 1 was 1mg.

The total dose of the compound of example 1 was 0.25mg.

In addition, it is preferable that the total dose of the pharmaceutical composition comprising the compound of example 1 or a pharmaceutically acceptable salt thereof is contained in one unit dose.

It is further preferred that the total dose of the pharmaceutical composition comprising the compound of example 1, or a pharmaceutically acceptable salt thereof, is contained in one unit dose, wherein the single unit dose is administered once per day.

It is also preferred that the total dose of the pharmaceutical composition comprising the compound of example 1, or a pharmaceutically acceptable salt thereof, is contained in two unit doses.

Preferably, the total dose of the pharmaceutical composition comprising the compound of example 1 or a pharmaceutically acceptable salt thereof is contained in two unit doses, each containing an equivalent amount of the compound of example 1.

It is further preferred that the total dose of the pharmaceutical composition comprising the compound of example 1, or a pharmaceutically acceptable salt thereof, is contained in two unit doses, wherein each dose is administered over the day.

It is further preferred that the total dose of the pharmaceutical composition comprising the compound of example 1 or a pharmaceutically acceptable salt thereof is comprised in two unit doses, each unit dose comprising an equal amount of the compound of example 1, wherein each dose is administered during a day, preferably at least 8 hours apart.

Example 3: expression and purification of engineered anti-N3 pGlu aβ antibodies

Antibodies to N3pGlu aβ are known in the art. For example, U.S. patent No. 8,679,498; U.S. patent No. 8,961,972; U.S. patent No. 10,647,759; and U.S. patent No. 11,078,261 (incorporated herein by reference in its entirety) discloses anti-N3 pGlu aβ antibodies, methods of making antibodies, antibody formulations, and methods of treating diseases such as alzheimer's disease with such antibodies. The amino acid sequences of exemplary anti-N3 pG aβ antibodies are provided in table 8 below.

Table 8: anti-N3 pGlu A.beta.antibodies SEQ ID NOs.

Antibodies to	Light chain	Heavy chain	LCVR	HCVR
					I	3	4	1	2
II	13	14	11	12

The anti-N3 pGlu aβ antibodies of the invention can be prepared and purified essentially as follows. For antibody I, the best predetermined HC was used: LC vector ratio or simultaneously encodes HC as set forth in SEQ ID NO:22 and LC are as set forth in SEQ ID NO:21, transiently or stably transfecting an appropriate host cell, e.g., HEK 293EBNA or CHO, with an antibody-secreting expression system. The clarified medium that has secreted the antibody is purified using any of a number of commonly used techniques. For example, the medium can be conveniently applied to a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column is washed to remove non-specific binding components. Bound antibody is eluted, for example, by a pH gradient (e.g., 0.1M sodium phosphate buffer pH6.8 to 0.1M sodium citrate buffer pH 2.5). Antibody fractions are detected, for example, by SDS-PAGE, and then pooled. Further purification is optional depending on the intended use. The antibodies can be concentrated and/or sterile filtered using conventional techniques. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion, hydrophobic interactions, ion exchange or hydroxyapatite chromatography. After these chromatographic steps, the purity of antibody I was greater than 99%. The product can be immediately frozen or lyophilized at-70 ℃.

Antibody II can be expressed and purified essentially as follows. Chinese hamster ovary cell line (CHO) was transfected by electroporation using a Glutamine Synthetase (GS) expression vector comprising a DNA sequence encoding the HC amino acid sequence of SEQ ID No. 24 and a DNA sequence encoding the LC amino acid sequence of SEQ ID No. 23. The expression vector encodes the SV Early (Simian Virus 40E) promoter and the GS gene. Following transfection, cells were selected in batches with 0-50. Mu. M L-methionine sulfoxide imine (MSX). The selected large number of cells or master wells are then scaled up in serum-free suspension culture for production. The clarified medium from which the antibodies have been secreted is applied to a Protein A affinity column which has been equilibrated with a compatible buffer, for example phosphate buffered saline (pH 7.4). The column was washed with 1M NaCl to remove non-specific binding components. Bound N3pGlu aβ antibodies are eluted, for example with sodium citrate at pH (about) 3.5, and the fractions are neutralized with 1M Tris buffer. The anti-N3 pGlu A beta antibody fractions are detected, for example by SDS-PAGE or analytical size exclusion chromatography and pooled. anti-N3 pGlu A.beta.antibody II was concentrated in PBS buffer pH7.4 or in 10mM sodium citrate buffer, 150mM NaCl at pH about 6. The final material may be sterile filtered using conventional techniques. The purity of the anti-N3 pGlu A.beta.antibody II was more than 95%. The anti-N3 pGlu Abeta antibody II of the present invention can be frozen immediately at-70℃or stored for several months at 4 ℃.

Example 4: binding affinity and kinetics of anti-N3 pGlu aβ antibodies.

The binding affinity and kinetics of the anti-N3 pGlu aβ antibodies of the invention (antibody I or antibody II) to pE 3-42A β peptide were measured by surface plasmon resonance using BIACORE ³⁰⁰⁰ (GE HEALTHCARE). By at least one ofImmobilized protein a on CMS chip captured anti-N3 pGlu aβ antibody and flowed pE 3-42A β peptide (2-fold serial dilutions to 3.125nM starting at 100 nM) to measure binding affinity. The experiment was performed in HBS-EP buffer (GE HEALTHCARE BR100669;10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4) at 25 ℃.

For each cycle, 5. Mu.L of antibody solution at a concentration of 10. Mu.g/mL was injected at a flow rate of 10. Mu.L/min to capture the antibody. mu.L of binding peptide was injected at 50. Mu.L/min and then dissociated for 10 minutes. The chip surface was regenerated by injecting 5. Mu.L glycine buffer pH1.5 at a flow rate of 10. Mu.L/mL. The data were fitted to a 1:1langmiur binding model to derive K _on、k_off and K _D was calculated. The following parameters (shown in table 9) were observed substantially as described above.

Table 9: binding affinity and kinetics.

Antibodies to	k_on(x10⁵1/Ms)	k_off(x10^-41/s)	K_D(nM)
				I	3.62	2.7	0.75
II	1.64E+03	6.98E-05	4.57

These data demonstrate that the antibodies of the invention bind pE 3-42A β.

Example 5: ex vivo target engagement

To determine ex vivo target engagement on brain sections from fixed PDAPP brains, immunohistochemical analysis was performed with exogenously added anti-N3 pGlu aβ antibodies of the invention (antibody I or antibody II). Cryostat series coronal sections from aged PDAPP mice (25 months old) were incubated with 20 μg/mL of an exemplary anti-N3 pGluA β antibody of the invention. The deposited plaques were visualized using a secondary HRP reagent specific for human IgG and DAB-Plus (DAKO). Biotinylated murine 3D6 antibody was used followed by Step-HRP secondary antibody as positive control. Positive control antibody (biotinylated 3D 6) labeled aβ deposited in large amounts in PDAPP hippocampus, anti-N3 pGlu aβ antibody (antibody I or antibody II) labeled a subset of deposits. These histological studies demonstrated that the anti-N3 pGlu aβ antibodies of the invention bind to the deposited aβ target in an ex vivo experiment.

Example 6: in vivo target engagement studies

The ability of the anti-N3 pGlu aβ antibodies of the present disclosure to cross the blood brain barrier and bind to deposited plaques in vivo was measured. Older PDAPP transgenic mice (18.5 to 32 months old) were intraperitoneally injected with anti-N3 pGlu aβ antibodies (e.g., antibodies I or II) or negative control IgG. Six mice per group received one 40mg/kg antibody injection on day 1 and day 3. Mice were sacrificed on day 6 and brains were collected for histochemical analysis to determine in vivo target engagement.

The extent of in vivo target engagement was quantified as the percentage of positive area for in vivo anti-N3 pGlu aβ antibody engagement normalized to the total plaque area (TE ratio) defined by exogenous control antibody immunostaining on sister sections. TE ratio was generated by measuring the percentage of area bound by the antibody and normalizing this value to the total percentage of area of possible targets (total deposition aβ was visualized by exogenous immunohistochemical visualization on sister sections using positive control antibody (3D 6).

Both anti-N3 pGlu aβ antibodies I and II were found to bind to the deposited plaques, essentially as described above. These results demonstrate that anti-N3 pGlu antibodies I and II can cross the blood brain barrier and engage the intended target of deposited aβ when administered peripherally.

Example 7: preparation of the OGA inhibitor 1- (2- (((3 r,5 s) -1- ((6-fluoro-2-methylbenzo [ d ] thiazol-5-yl) methyl) -5-methylpyrrolidin-3-yl) oxy) -5, 7-dihydro-6H-pyrrolo [3,4-b ] pyridin-6-yl) ethan-1-one.

This compound and its synthesis are disclosed in U.S. patent No. US10,752,362, the entire contents of which are incorporated herein by reference.

To a solution of 6-fluoro-2-methyl-1, 3-benzothiazole-5-carbaldehyde (0.19 g,0.95 mmol) and 1- (2- (((3R, 5S) -5-methylpyrrolidin-3-yl) oxy) -5, 7-dihydro-6H-pyrrolo [3,4-b ] pyridin-6-yl) ethan-1-one hydrochloride (0.28 g,0.94 mmol) in DCM (9 mL) was added DIPEA (0.45 mL,2.6 mmol). The resulting solution was stirred at RT for 40 min. To this solution was added sodium triacetoxyborohydride (NaBH (OAc) ₃, 0.65g,3.04 mmol). The resulting solution was stirred at RT for 17 hours. The reaction mixture was slowly quenched with saturated aqueous NaHCO ₃ (5 mL). The aqueous layer was extracted with DCM (2X 5 mL). The combined organic extracts were dried over MgSO ₄, filtered and concentrated under reduced pressure. The resulting residue was dissolved in DCM and purified by flash chromatography on silica gel, gradient elution with 40-100% acetone in hexane, affording the title compound (0.27 g,65% yield) after evaporation of the solvent of the desired chromatographic fraction. ES/MS M/z 441 (M+H); [ α ] _D ²⁰ = +101.4 ° (c=0.2, meoh).

Alternative preparation of the OGA inhibitor 1- (2- (((3 r,5 s) -1- ((6-fluoro-2-methylbenzo [ d ] thiazol-5-yl) methyl) -5-methylpyrrolidin-3-yl) oxy) -5, 7-dihydro-6H-pyrrolo [3,4-b ] pyridin-6-yl) ethan-1-one: 4-methylbenzenesulfonic acid; preparation of (3R, 5S) -5-methylpyrrolidin-3-ol.

To the flask were added (2S, 4R) -4-hydroxy-2-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester (53.0 g,263 mmol) and 2-propanol (265 mL) at room temperature. The mixture was stirred at room temperature (internal temperature 20 ℃) and p-toluenesulfonic acid monohydrate (60.1 g,316 mmol) was added in one portion. The reaction mixture was stirred overnight in a 62 ℃ heating block, then cooled to room temperature and concentrated to a total volume of about 150 mL. The mixture was diluted with methyl tert-butyl ether (MTBE, 530 mL) and the mixture was vigorously stirred at room temperature for 30min, then filtered under a stream of N ₂. The filtered solid is dried in vacuum at 40 ℃ for 2 hours to obtain 4-methylbenzenesulfonic acid; (3R, 5S) -5-methylpyrrolidin-3-ol (67.6 g,93% yield) as a white solid. ES/MS m/z:102 (M+H).

Preparation of (3R, 5S) -1- [ (6-fluoro-2-methyl-1, 3-benzothiazol-5-yl) methyl ] -5-methyl-pyrrolidin-3-ol.

Adding 4-methylbenzenesulfonic acid into a flask at room temperature; (3R, 5S) -5-methylpyrrolidin-3-ol (61.9 g,226 mmol), etOAc (850 mL) and 6-fluoro-2-methyl-1, 3-benzothiazole-5-carbaldehyde (42.5 g,216 mmol). The reaction mixture was stirred in an ice water bath (internal temperature 3 ℃) and triethylamine (60.1 mL,431 mmol) was added in one portion. The reaction mixture was stirred in an ice water bath for 30 minutes, then sodium triacetoxyborohydride (91.4 g,431 mmol) was added in one portion. The reaction mixture was stirred in an ice-water bath for 10 minutes and then at room temperature for 2 hours (internal temperature 20 ℃). The reaction mixture was stirred in an ice water bath and 15% aqueous khso ₄ (650 mL) was added over 5 minutes, keeping the internal temperature below 15 ℃ during the addition. The mixture was stirred vigorously at room temperature for 1 hour, then saturated aqueous citric acid (100 mL) was added and the mixture was stirred at room temperature for 5 minutes, then the layers were separated. The aqueous layer was washed with EtOAc (400 mL) and then stirred in an ice-water bath, and solid Na ₂CO₃ (80 g) was added in portions over 10 minutes with vigorous stirring until ph=10 (measured by pH paper). The aqueous layer was then extracted with EtOAc (3X 400 mL). The combined organics were dried over Na ₂SO₄ and concentrated to give a residue, which was crushed to a fine powder using a pestle and mortar, then combined with 25% mtbe/heptane (280 mL). The mixture was vigorously stirred in a 45 ℃ heating block for 1 hour, then at room temperature for 1 hour, and then filtered to give a first filtered solid. The filtrate was concentrated, then the residue was combined with 25% mtbe/heptane (40 mL) and the mixture was vigorously stirred at room temperature for 30 minutes, then filtered to give a second filtered solid. The first and second filtered solids were combined and the mixture was triturated with a spatula and then dried overnight under vacuum at room temperature to give (3 r,5 s) -1- [ (6-fluoro-2-methyl-1, 3-benzothiazol-5-yl) methyl ] -5-methyl-pyrrolidin-3-ol (53.3 g,87% yield) as a cream colored solid. ES/MS m/z:281 (M+H).

Alternative preparation of the OGA inhibitor 1- (2- (((3 r,5 s) -1- ((6-fluoro-2-methylbenzo [ d ] thiazol-5-yl) methyl) -5-methylpyrrolidin-3-yl) oxy) -5, 7-dihydro-6H-pyrrolo [3,4-b ] pyridin-6-yl) ethan-1-one.

To the flask was added (3R, 5S) -1- [ (6-fluoro-2-methyl-1, 3-benzothiazol-5-yl) methyl ] -5-methyl-pyrrolidin-3-ol (26.9 g,95.0 mmol), 1- (2-chloro-5, 7-dihydropyrrolo [3,4-b ] pyridin-6-yl) ethanone (22.1 g,109 mmol), cesium carbonate (92.8 g, 284 mmol), morDalPhos (1.76 g,3.80 mmol), palladium (II) chloride (pi-cinnamyl) dimer (984 mg,1.90 mmol) and toluene (538 mL) at room temperature. N ₂ gas was bubbled into the mixture with stirring at room temperature for 30 minutes, and the reaction mixture was stirred in a 86℃heating block overnight (internal temperature 80 ℃). The reaction mixture was cooled to room temperature and diluted with EtOAc (269 mL) and celite (27 g) was added. The mixture was stirred at room temperature for 5min, then filtered through celite, washing with EtOAc (200 mL). The filtrate was concentrated to give a residue, which was dissolved in EtOAc (100 mL) and the mixture was passed through a short pad of silica gel (300 g), eluting with EtOAc (2L), then 20% isopropanol/EtOAc (IPA/EtOAc, 2L). The IPA/EtOAc fraction was concentrated to give a residue which was dried at room temperature under vacuum for 1 hour to give the title compound (42.1 g,88% yield, 88% mass purity) as a pale brown foam.

The foam was combined with another batch of material of similar purity at room temperature, and the combined material (46.0 g,92.3 mmol) was combined with MTBE (230 mL) and heptane (230 mL). The mixture was vigorously stirred in a 45 ℃ heating block for 1 hour, then stirred at room temperature for 30 minutes, and then filtered. The filtered solid was combined with EtOAc (400 mL) and addedThiol (40 g). The mixture was stirred on a rotary evaporator at room temperature for 1 hour and then filtered. The filtrate was concentrated to give a residue, which was mixed with 25% etoac/heptane (400 mL) and the mixture was vigorously stirred in a 50 ℃ heated block for 1 hour, then stirred at room temperature for 10 minutes, then filtered, leaving a first filtrate. The filtered solids were combined with 35% etoac/heptane (400 mL) and the mixture was vigorously stirred in a 50 ℃ heated block for 1 hour, then stirred at room temperature for 10 minutes, then filtered, leaving a second filtrate. The filtered solid was combined with EtOAc (500 mL) and 15% aqueous khso ₄ (500 mL). The mixture was stirred vigorously at room temperature for 15 minutes, then transferred to a separatory funnel and the layers separated, leaving a residue layer in the organics. The organic layer was further extracted with 15% aqueous KHSO ₄ (100 mL) leaving a residue layer in the organics. The residue layer was removed from the organics and diluted with CH ₂Cl₂ (100 mL) and 15% aqueous KHSO ₄ (100 mL) and the layers separated. The combined aqueous layers were stirred in an ice-water bath and solid Na ₂CO₃ (100 g) (pH 10 measured by pH paper) was added in portions over 5 minutes with stirring. The mixture was extracted with CH ₂Cl₂ (2 x 500 mL) and the combined organics were dried over Na ₂SO₄ and concentrated to give a first crude product. The first and second filtrates from filtration were combined and concentrated, then the residue was combined with EtOAc (100 mL) and 15% aqueous khso ₄ (100 mL). The mixture was stirred vigorously at room temperature for 15 minutes, then transferred to a separatory funnel and the layers separated. The aqueous layer was stirred in an ice-water bath, and solid Na ₂CO₃ (15 g) (pH 10 measured by pH paper) was added in portions over 5 minutes with stirring. The mixture was extracted with CH ₂Cl₂ (2 x 100 mL) and the combined organics were dried over Na ₂SO₄ and concentrated to give a residue which was combined with 25% etoac/heptane (80 mL) and the mixture was vigorously stirred in a 50 ℃ heating block for 30 min, then at room temperature for 10 min, then filtered to give a second crude product. The two batches of crude product were combined with 25% etoac/heptane (400 mL) and the mixture was vigorously stirred in a 50 ℃ heating block for 30 min, then at room temperature for 10 min, then filtered. The filtered solid was dried in vacuo at room temperature for 3 days to give the final title compound (37.4 g,90% yield) as a white solid. ES/MS M/z 441 (M+H). Optical rotation [ α ] D ₂₀ = +104.0 ° (c=0.2, meoh).

The compound of example 7 was tested in vitro for human OGA enzyme assay substantially as described above and showed IC ₅₀ of 0.214nm±0.037 (n=4). This data demonstrates that the compound of example 7 inhibits OGA enzyme activity in vitro. The compound of example 7 was tested to measure inhibition of OGA enzyme activity substantially as per the whole cell assay described above and exhibited an IC ₅₀ of 70.5nm±0.002 (n=2). This data demonstrates that the compound of example 7 inhibits OGA enzyme activity in a cellular assay.

Example 8: in vivo combinatorial research

The following examples illustrate how a study was designed to demonstrate that the combination of the OGA inhibitors of the present disclosure with the anti-N3 pGlu aβ antibodies of the invention can be used to treat diseases characterized by amyloid deposition and/or abnormal tau aggregation, such as AD. However, it is to be understood that the following description is set forth by way of illustration and not limitation, and that various modifications may be made by one of ordinary skill in the art.

To assess the effect of exemplary OGA inhibitors on tau hyperphosphorylation and reduced aggregation, and of exemplary anti-N3 pGlu aβ antibodies on reduced amyloid β deposition, in the combination therapies described herein, delay in disease progression can be assessed by biomarker and/or cognitive and functional decline assessment using a validated rating scale.

Patients can be divided into treatment groups consisting of a double blind placebo group and a combination treatment group. The combination treatment group is administered an effective amount of an OGA inhibitor in combination with an effective amount of an anti-N3 pGlu aβ antibody. The single agent treatment group (the same OGA inhibitor single agent treatment group as the OGA inhibitor dose in the combination group; the same anti-N3 pGlu aβ antibody single agent treatment group as the anti-N3 pGlu aβ antibody dose in the combination treatment group) may be included to further elucidate the contribution of each individual molecule to the disease change. Furthermore, the characteristics of the treatment group may be based on the diagnosis of pre-clinical or clinical AD, or on the diagnosis of patients (although without symptoms of AD) having mutations in the genes responsible for AD disease. For example, the group may include one or more of the following: (a) positive for asymptomatic but AD-causing gene mutations; (b) a precursor phase AD; (c) mild AD dementia; (d) moderate AD dementia; (e) severe AD dementia. Each treatment group may receive a corresponding treatment (e.g., once every four weeks for anti-N3 pGlu aβ antibody treatment and once daily for OGA inhibitor treatment) for a treatment period of 9 months to 18 months.

After the end of the treatment period, AD neurodegeneration may be assessed by one or more of the following biomarker assessments: (a) amyloid PET imaging; (b) Phosphorylated tau (P-tau; phosphorylated at threonine 181 or 217); (c) Tau PET imaging (assessing NFT accumulation); (d) volumetric MRI (neuroanatomical atrophy assessment); (e) FDG-PEG PET imaging (hypometabolic assessment); (f) Fluobetapirtine perfusion PET imaging (metabolic hypo assessment); (g) CSF tau concentration (neurodegeneration assessment); and/or (i) CSF phosphorylation Tau concentration (neurodegeneration assessment). In addition, one or more validated ratings scales, such as ADAS-cog, MMSE, CDR-SB, ADCS-ADL, and Functional Activity Questionnaire (FAQ), that evaluate cognitive and functional decline for each treatment group may be applied.

This study may show that the combination treatment of the OGA inhibitors of the invention and the anti-N3 pGlu aβ antibodies of the invention may lead to a reduction of aβ plaques and limit tau hyperphosphorylation and neuro-cohesion to pathological tau, such as NFT, for use in the treatment of diseases such as AD.

Sequence(s)

LCVR of sequence ID NO. 1 antibody I

HCVR of sequence ID No. 2 antibody I

LC of sequence ID NO 3 antibody I

HC of sequence ID NO.4 antibody I

LCDR1 of sequence ID No. 5 antibody I

LCDR2 of sequence ID NO 6 antibody I

LCDR3 of sequence ID NO 7 antibody I

HCDR1 of sequence ID NO 8 antibody I

HCDR2 of sequence ID NO 9 antibody I

HCDR3 of antibody I of sequence ID NO 10

LCVR of sequence ID NO 11 antibody II

HCVR of SEQ ID NO. 12 antibody II

LC of sequence ID NO 13 antibody II

HC of sequence ID No. 14 antibody II

LCDR1 of sequence ID NO 15 antibody II

LCDR2 of sequence ID NO 16 antibody II

LCDR3 of sequence ID NO 17 antibody II

HCDR1 of sequence ID NO 18 antibody II

HCDR2 of sequence ID NO 19 antibody II

HCDR3 of sequence ID NO 20 antibody II

LC DNA sequence of sequence ID NO. 21 antibody I

HC DNA sequence of sequence ID NO. 22 antibody I

LC DNA sequence of sequence ID NO 23 antibody II

HC DNA sequence of sequence ID NO. 24 antibody II

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Claims

1. A method of treating a patient suffering from a disorder characterized by amyloid beta (Abeta) deposition comprising administering to the patient in need thereof an effective amount of an anti-N3 pG Abeta antibody in combination with an effective amount of an OGA inhibitor,

Wherein the OGA inhibitor is a compound of the formula:

Or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the disease characterized by amyloid β (aβ) deposition is selected from the group consisting of preclinical Alzheimer's Disease (AD), clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, severe AD dementia, down's syndrome, clinical cerebral amyloid angiopathy, and preclinical cerebral amyloid angiopathy.

3. The method of claim 1, wherein the methyl group at the 2-position of the OGA inhibitor is in the cis configuration relative to the oxygen at the 4-position of the piperidine ring:

4. The method of claim 3, wherein the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2 s,4 s) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide.

5. The method of claim 4, wherein the OGA inhibitor is crystalline.

6. The method of claim 5, wherein the compound is characterized by a combination of a peak having a diffraction angle 2- θ of 12.1 ° and one or more peaks selected from 15.3 °, 21.6 °, 22.2 °, 22.7 °, 23.5 °, 24.3 °, and 26.8 ° in an X-ray powder diffraction spectrum, the diffraction angle error being 0.2 °.

7. The method of claim 1, wherein the anti-N3 pga beta antibody comprises a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR), wherein the LCVR comprises Complementarity Determining Regions (CDRs) LCDR1, LCDR2 and LCDR3 and the HCVR comprises CDRs HCDR1, HCDR2 and HCDR3, wherein:

i) The amino acid sequence of LCDR1 is given by SEQ ID NO.5, the amino acid sequence of LCDR2 is given by SEQ ID NO.6, the amino acid sequence of LCDR3 is given by SEQ ID NO.7, the amino acid sequence of HCDR1 is given by SEQ ID NO.8, the amino acid sequence of HCDR2 is given by SEQ ID NO.9 and the amino acid sequence of HCDR3 is given by SEQ ID NO. 10; or alternatively

Ii) the amino acid sequence of LCDR1 is given by SEQ ID NO.15, the amino acid sequence of LCDR2 is given by SEQ ID NO.16, the amino acid sequence of LCDR3 is given by SEQ ID NO.17, the amino acid sequence of HCDR1 is given by SEQ ID NO.18, the amino acid sequence of HCDR2 is given by SEQ ID NO.19 and the amino acid sequence of HCDR3 is given by SEQ ID NO. 20.

8. The method of claim 7, wherein the anti-N3 pG aβ antibody comprises a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR), wherein:

i) The amino acid sequence of LCVR is given by SEQ ID NO. 1 and the amino acid sequence of HCVR is given by SEQ ID NO. 2; or alternatively

Ii) the amino acid sequence of LCVR is given by SEQ ID NO. 11 and the amino acid sequence of HCVR is given by SEQ ID NO. 12.

9. The method of claim 8, wherein the anti-N3 pG aβ antibody comprises a Light Chain (LC) and a Heavy Chain (HC), wherein:

i) The amino acid sequence of LC is given by SEQ ID NO. 3 and the amino acid sequence of HC is given by SEQ ID NO. 4; or alternatively

Ii) the amino acid sequence of LC is given by SEQ ID NO. 13 and the amino acid sequence of HC is given by SEQ ID NO. 14.

10. A method of treating a patient suffering from a disorder characterized by amyloid β (aβ) deposition, comprising administering to a patient in need of such treatment an effective amount of an anti-N3 pgaβ antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of the formula:

Or a pharmaceutically acceptable salt thereof.

11. The method of claim 10, wherein the disease characterized by amyloid β (aβ) deposition is selected from preclinical alzheimer's disease, clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, severe AD dementia, down's syndrome, clinical cerebral amyloid angiopathy, or preclinical cerebral amyloid angiopathy.

12. The method of claim 10, wherein the methyl group at the 2-position of the OGA inhibitor is in the cis configuration relative to the oxygen at the 4-position of the piperidine ring:

13. the method of claim 10, wherein the methyl group at the 2-position of the OGA inhibitor is in the trans configuration relative to the oxygen at the 4-position of the piperidine ring:

14. The method of claim 12, wherein the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2 s,4 s) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide.

15. The method of claim 13, wherein the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2 s,4 s) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide.

16. The method of claim 14, wherein the OGA inhibitor is crystalline.

17. The method of claim 15, wherein the OGA inhibitor is characterized by a combination of a peak having a diffraction angle 2- θ of 13.5 ° and one or more peaks selected from 5.8 °, 13.0 °, 14.3 °, 17.5 °, 20.4 °, 21.4 ° and 22.2 ° in an X-ray powder diffraction spectrum, with a diffraction angle error of 0.2 °.

18. The method of claim 10, wherein the anti-N3 pga beta antibody comprises a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR), wherein the LCVR comprises Complementarity Determining Regions (CDRs) LCDR1, LCDR2 and LCDR3 and the HCVR comprises CDRs HCDR1, HCDR2 and HCDR3, wherein:

19. The method of claim 18, wherein the anti-N3 pG aβ antibody comprises a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR), wherein:

20. The method of claim 19, wherein the anti-N3 pG aβ antibody comprises a Light Chain (LC) and a Heavy Chain (HC), wherein:

21. A method of treating a patient suffering from a disorder characterized by amyloid beta (Abeta) deposition and abnormal tau aggregation comprising administering to the patient in need thereof an effective amount of an anti-N3 pG Abeta antibody in combination with an effective amount of an OGA inhibitor,

Wherein the OGA inhibitor is a compound of the formula:

Or a pharmaceutically acceptable salt thereof.

22. The method of claim 21, wherein the disease characterized by amyloid β (aβ) deposition and abnormal tau aggregation is selected from preclinical Alzheimer's Disease (AD), clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, severe AD dementia, down's syndrome, clinical cerebral amyloid angiopathy, and preclinical cerebral amyloid angiopathy.

23. The method of claim 21, wherein the methyl group at the 2-position of the OGA inhibitor is in the cis configuration relative to the oxygen at the 4-position of the piperidine ring:

24. The method of claim 23, wherein the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2 s,4 s) -2-methyl-4- [ (5-methyl-1, 2, 4-oxadiazol-3-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide.

25. The method of claim 24, wherein the OGA inhibitor is crystalline.

26. The method of claim 25, wherein the compound is characterized by a combination of a peak having a diffraction angle 2- θ of 12.1 ° and one or more peaks selected from 15.3 °, 21.6 °, 22.2 °, 22.7 °, 23.5 °, 24.3 °, and 26.8 ° in an X-ray powder diffraction spectrum, the diffraction angle error being 0.2 °.

27. The method of claim 21, wherein the anti-N3 pga beta antibody comprises a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR), wherein the LCVR comprises Complementarity Determining Regions (CDRs) LCDR1, LCDR2 and LCDR3 and the HCVR comprises CDRs HCDR1, HCDR2 and HCDR3, wherein:

28. The method of claim 27, wherein the anti-N3 pG aβ antibody comprises a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR), wherein:

29. The method of claim 28, wherein the anti-N3 pG aβ antibody comprises a Light Chain (LC) and a Heavy Chain (HC), wherein:

30. A method of treating a patient suffering from a disorder characterized by amyloid β (aβ) deposition and abnormal tau aggregation, comprising administering to a patient in need of such treatment an effective amount of an anti-N3 pG aβ antibody in combination with an effective amount of an OGA inhibitor, wherein the OGA inhibitor is a compound of the formula:

Or a pharmaceutically acceptable salt thereof.

31. The method of claim 30, wherein the disease characterized by amyloid β (aβ) deposition and abnormal tau aggregation is selected from the group consisting of preclinical alzheimer's disease, clinical AD, prodromal AD, mild AD dementia, moderate AD dementia, severe AD dementia, down's syndrome, clinical cerebral amyloid angiopathy, and preclinical cerebral amyloid angiopathy.

32. The method of claim 30, wherein the methyl group at the 2-position of the OGA inhibitor is in the cis configuration relative to the oxygen at the 4-position of the piperidine ring:

33. the method of claim 30, wherein the methyl group at the 2-position of the OGA inhibitor is in the trans configuration relative to the oxygen at the 4-position of the piperidine ring:

34. The method of claim 32, wherein the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2 s,4 s) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide.

35. The method of claim 33, wherein the OGA inhibitor is N- [ 4-fluoro-5- [ [ (2 s,4 s) -2-methyl-4- [ (5-methyl-1, 3, 4-oxadiazol-2-yl) methoxy ] -1-piperidinyl ] methyl ] thiazol-2-yl ] acetamide.

36. The method of claim 34, wherein the OGA inhibitor is crystalline.

37. The method of claim 35, wherein the OGA inhibitor is characterized by a combination of a peak having a diffraction angle 2- θ of 13.5 ° and one or more peaks selected from 5.8 °, 13.0 °, 14.3 °, 17.5 °, 20.4 °, 21.4 ° and 22.2 ° in an X-ray powder diffraction spectrum, with a diffraction angle error of 0.2 °.

38. The method of claim 30, wherein the anti-N3 pga beta antibody comprises a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR), wherein the LCVR comprises Complementarity Determining Regions (CDRs) LCDR1, LCDR2 and LCDR3 and the HCVR comprises CDRs HCDR1, HCDR2 and HCDR3, wherein:

39. The method of claim 38, wherein the anti-N3 pG aβ antibody comprises a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR), wherein:

40. The method of claim 39, wherein the anti-N3 pG aβ antibody comprises a Light Chain (LC) and a Heavy Chain (HC), wherein: