NZ754849B2 - N-[4-fluoro-5-[[(2s,4s)-2-methyl-4-[(5-methyl-1,2,4-oxadiazol-3-yl)methoxy]-1-piperidyl]methyl]thiazol-2-yl]acetamide as oga inhibitor - Google Patents
N-[4-fluoro-5-[[(2s,4s)-2-methyl-4-[(5-methyl-1,2,4-oxadiazol-3-yl)methoxy]-1-piperidyl]methyl]thiazol-2-yl]acetamide as oga inhibitor Download PDFInfo
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- NZ754849B2 NZ754849B2 NZ754849A NZ75484918A NZ754849B2 NZ 754849 B2 NZ754849 B2 NZ 754849B2 NZ 754849 A NZ754849 A NZ 754849A NZ 75484918 A NZ75484918 A NZ 75484918A NZ 754849 B2 NZ754849 B2 NZ 754849B2
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- NZ
- New Zealand
- Prior art keywords
- compound
- methyl
- pharmaceutically acceptable
- acceptable salt
- mixture
- Prior art date
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- 239000003112 inhibitor Substances 0.000 title description 7
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 1
- 229960002303 citric acid monohydrate Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000005712 crystallization Effects 0.000 description 1
- 238000010192 crystallographic characterization Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940042399 direct acting antivirals Protease inhibitors Drugs 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 229940079593 drugs Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000003821 enantio-separation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000001747 exhibiting Effects 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000008098 formaldehyde solution Substances 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 239000003316 glycosidase inhibitor Substances 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- IZZWJPQHPPRVLP-UHFFFAOYSA-N hexane;2-methoxy-2-methylpropane Chemical compound CCCCCC.COC(C)(C)C IZZWJPQHPPRVLP-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000000670 limiting Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000002609 media Substances 0.000 description 1
- GRVDJDISBSALJP-UHFFFAOYSA-N methyloxidanyl Chemical compound [O]C GRVDJDISBSALJP-UHFFFAOYSA-N 0.000 description 1
- QDHHCQZDFGDHMP-UHFFFAOYSA-N monochloramine Chemical compound ClN QDHHCQZDFGDHMP-UHFFFAOYSA-N 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000001537 neural Effects 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000010502 orange oil Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N oxygen atom Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000036961 partial Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- RJUAEBLXGFKZMS-UHFFFAOYSA-N piperidin-1-ylmethanol Chemical compound OCN1CCCCC1 RJUAEBLXGFKZMS-UHFFFAOYSA-N 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 230000001681 protective Effects 0.000 description 1
- 238000001711 protein immunostaining Methods 0.000 description 1
- 229910052904 quartz Inorganic materials 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000010956 selective crystallization Methods 0.000 description 1
- 231100000486 side effect Toxicity 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 229940001584 sodium metabisulfite Drugs 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 150000004685 tetrahydrates Chemical class 0.000 description 1
- GTDKXDWWMOMSFL-UHFFFAOYSA-M tetramethylazanium;fluoride Chemical compound [F-].C[N+](C)(C)C GTDKXDWWMOMSFL-UHFFFAOYSA-M 0.000 description 1
- 201000007023 thrombotic thrombocytopenic purpura Diseases 0.000 description 1
- 230000001052 transient Effects 0.000 description 1
- 238000001665 trituration Methods 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
- 229940102001 zinc bromide Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/09—Geometrical isomers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/13—Crystalline forms, e.g. polymorphs
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
Abstract
The present invention provides a compound of Formula (I): or a pharmaceutically acceptable salt thereof, and the use of compounds of Formula (I) for treatment of neurodegenerative diseases and disorders, such as Alzheimer's disease.
Description
N-[4-FLUORO[[(2S,4S)METHYL[(5-METHYL-1,2,4-OXADIAZOL
YL)METHOXY]PIPERIDYL]METHYL]THIAZOLYL]ACETAMIDE AS OGA
INHIBITOR
The present invention relates to novel 5-methyl-1,2,4-oxadiazolyl compounds,
to pharmaceutical compositions comprising the compounds, and to intermediates and
processes useful in the synthesis of the compounds. Methods of using the compounds to
treat physiological disorders are described.
The present invention is in the field of treatment of Alzheimer’s disease,
progressive supranuclear palsy (PSP) and other diseases and disorders involving
tau-mediated neurodegeneration, known collectively as tauopathies.
Alzheimer’s disease is a devastating neurodegenerative disorder that affects
millions of patients worldwide. In view of the currently approved agents on the market
which afford only transient, symptomatic benefits to the patient, there is a significant
unmet need in the treatment of Alzheimer’s disease.
The oligomerization of the microtubule-associated protein tau into filamentous
structures such as paired helical filaments (PHFs) and straight or twisted filaments, which
give rise to neurofibrillary tangles (NFTs) and neuropil threads (NTs), is one of the
defining pathological features of Alzheimer’s disease and other tauopathies. The number
of NFTs in the brains of individuals with Alzheimer’s disease has been found to correlate
closely with the severity of the disease, suggesting 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 correlate with disease duration in PSP;
cases with a more aggressive disease course have a higher tau burden than cases with a
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 hyperphosphorylation
and aggregation into pathological tau for the treatment of Alzheimer’s disease and related
tau-mediated neurodegeneration disorders. Specifically, the OGA inhibitor Thiamet-G
has been linked in slowing motor neuron loss in the JNPL3 tau mouse model (Yuzwa et
al., Nat Chem Biol, 8, 393-399 (2012)) and to a reduction in tau pathology and dystrophic
neurites in the Tg4510 tau mouse model (Graham et al., Neuropharmacology, 79, 307-
313 (2014)). Accordingly, OGA inhibitors are recognized as a valid therapeutic approach
to reduce the accumulation of hyperphosphorylated, pathological forms of tau, such as
NFTs and NTs.
U.S. Patent No. 9,120,781 discloses hexahydrobenzooxazole and
hexahydrobenzothiazole derivatives which possess OGA inhibitory activity and are
further disclosed as useful in treating diseases and disorders related to deficiency or
overexpression of OGA, and/or accumulation or deficiency of 2-acetamidodeoxy-5ß-
D-glucopyranoside (O-GlcNAc). In addition, US 2016/0031871 discloses certain
glycosidase inhibitors for treating Alzheimer’s disease.
OGA inhibitors that are brain penetrant are desired to provide treatments for
tau-mediated neurodegeneration disorders, such as Alzheimer’s disease and PSP. The
present invention provides certain novel compounds that are inhibitors of OGA.
Accordingly, the present invention provides a compound of Formula I:
Formula I
or a pharmaceutically acceptable salt thereof.
In addition, the present invention provides a compound of Formula Ia:
Formula Ia
or a pharmaceutically acceptable salt thereof.
Described herein is a method of treating Alzheimer’s disease in a patient in need
of such treatment, comprising administering to the patient an effective amount of a
compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof.
Described herein is a method of treating the progression of mild cognitive
impairment to Alzheimer’s disease in a patient in need of such treatment, comprising
administering to the patient an effective amount of a compound of Formulas I or Ia, or a
pharmaceutically acceptable salt thereof.
Described herein is a method of treating progressive supranuclear palsy in a
patient in need of such treatment, comprising administering to the patient an effective
amount of a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof.
Described herein is a method of treating tau-mediated neurodegenerative disorders in a
patient, comprising administering to a patient in need of such treatment an effective
amount of a compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof.
Furthermore, this invention provides a compound of Formulas I or Ia, or a
pharmaceutically acceptable salt thereof for use in therapy, in particular for use in the
treatment of Alzheimer’s disease or for use in preventing the progression of mild
cognitive impairment to Alzheimer’s disease. In addition, this invention provides a
compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof for use in the
treatment of progressive supranuclear palsy. The invention also provides a compound of
Formulas I or Ia, or a pharmaceutically acceptable salt thereof for use in treating tau-
mediated neurodegenerative disorders.
Even furthermore, this invention provides the use of a compound of Formulas I or
Ia, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for
the treatment of Alzheimer’s disease or for preventing the progression of mild cognitive
impairment to Alzheimer’s disease. In addition, this invention provides the use of a
compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof, for the
manufacture of a medicament for the treatment of progressive supranuclear palsy. The
invention also provides the use of a compound of Formulas I or Ia, or a pharmaceutically
acceptable salt thereof, for the manufacture of a medicament for treating tau-mediated
neurodegenerative disorders.
The invention further provides a pharmaceutical composition, comprising a
compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof, with one or
more pharmaceutically acceptable carriers, diluents, or excipients. The invention further
provides a process for preparing a pharmaceutical composition, comprising admixing a
compound of Formulas I or Ia, or a pharmaceutically acceptable salt thereof, with one or
more pharmaceutically acceptable carriers, diluents, or excipients. This invention also
relates to novel intermediates and processes for the synthesis of the compounds of
Formulas I and Ia.
Mild cognitive impairment has been defined as a potential prodromal phase of
dementia associated with Alzheimer’s disease based on clinical presentation and on
progression of patients exhibiting mild cognitive impairment to Alzheimer’s dementia
over time. The term “preventing the progression of mild cognitive impairment to
Alzheimer’s disease” includes restraining, slowing, stopping, or reversing the progression
of mild cognitive impairment to Alzheimer’s disease in a patient.
As used herein, the terms “treating” or “to treat” includes restraining, slowing,
stopping, or reversing the progression or severity of an existing symptom or disorder.
As used herein, the term "patient" refers to a human.
As used herein, the term “effective amount” refers to the amount or dose of
compound of the invention, or a pharmaceutically acceptable salt thereof which, upon
single or multiple dose administration to the patient, provides the desired effect in the
patient under diagnosis or treatment.
An effective amount can be readily determined by one skilled in the art by the use
of known techniques and by observing results obtained under analogous circumstances.
In determining the effective amount for a patient, a number of factors are considered,
including, but not limited to: the species of patient; its size, age, and general health; the
specific disease or disorder involved; the degree of or involvement or the severity of the
disease or disorder; the response of the individual patient; the particular compound
administered; the mode of administration; the bioavailability characteristics of the
preparation administered; the dose regimen selected; the use of concomitant medication;
and other relevant circumstances.
The compounds of the present invention are generally effective over a wide
dosage range. For example, dosages per day normally fall within the range of about 0.1
to about 15 mg/kg of body weight. In some instances dosage levels below the lower limit
of the aforesaid range may be more than adequate, while in other cases still larger doses
may be employed with acceptable side effects, and therefore the above dosage range is
not intended to limit the scope of the invention in any way.
The compounds of the present invention are preferably formulated as
pharmaceutical compositions administered by any route which makes the compound
bioavailable, including oral and transdermal routes. Most preferably, such compositions
are for oral administration. Such pharmaceutical compositions and processes for
preparing same are well known in the art (See, e.g., Remington: The Science and Practice
of Pharmacy, L.V. Allen, Editor, 22 Edition, Pharmaceutical Press, 2012).
The compounds of Formulas I and Ia, or pharmaceutically acceptable salts thereof
are particularly useful in the treatment methods described herein, but certain
configurations are preferred. The following paragraphs describe such preferred
configurations. It will be understood that these preferences are applicable both to the
treatment methods described herein and to the compounds of the invention.
Compounds of the present invention include:
Formula Ia
Formula Ib
Formula Ic
Formula Id
and pharmaceutically acceptable salts thereof.
The compound of Formula I wherein the methyl and oxygen substituents on the
piperidine ring are in the cis or trans configuration, or pharmaceutically acceptable salt
thereof, are included within the scope of the invention, with the cis configuration being
preferred. For example, one of ordinary skill in the art will appreciate that the methyl at
position 2 is in the cis configuration relative to the oxygen at position 4 as shown in
Scheme A below:
Scheme A
Formula Ia
Formula Ic
In addition, one of ordinary skill in the art will appreciate that the methyl at
position 2 is in the trans configuration relative to the oxygen at position 4 as shown in
Scheme B below:
Scheme B
Formula Ib
Formula Id
Compounds wherein the chiral center at position 2 of piperidine ring is in the S-
configuration are further preferred. Although the present invention contemplates all
individual enantiomers and diasteromers, as well as mixtures of the enantiomers of said
compounds, including racemates, the compounds with the absolute configuration as set
forth below are particularly preferred:
N-[4-fluoro[[(2S,4S)methyl[(5-methyl-1,2,4-oxadiazolyl)methoxy]
piperidyl]methyl]thiazolyl]acetamide, and pharmaceutically acceptable salts thereof;
N-[4-fluoro[[(2S,4S)methyl[(5-methyl-1,2,4-oxadiazolyl)methoxy]
piperidyl]methyl]thiazolyl]acetamide are particularly preferred.
The crystalline form of N-[4-fluoro[[(2S,4S)methyl[(5-methyl-1,2,4-
oxadiazolyl)methoxy]piperidyl]methyl]thiazolyl]acetamide is especially
preferred. The crystalline form of N-[4-fluoro[[(2S,4S)methyl[(5-methyl-1,2,4-
oxadiazolyl)methoxy]piperidyl]methyl]thiazolyl]acetamide which is
characterized by a peak in the X-ray powder diffraction spectrum at diffraction angle 2-
theta of 12.1° in combination with one or more peaks selected from the group consisting
of 15.3°, 21.6°, 22.2°, 22.7°, 23.5°, 24.3°,and 26.8°, with a tolerance for the diffraction
angles of 0.2 degrees, is further preferred.
Individual isomers, enantiomers, and diastereomers may be separated or resolved
by one of ordinary skill in the art at any convenient point in the synthesis of compounds
of the invention, by methods such as selective crystallization techniques or chiral
chromatography (See for example, 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).
A pharmaceutically acceptable salt of the compounds of the invention can be
formed, for example, by reaction of an appropriate free base of a compound of the
invention and an appropriate pharmaceutically acceptable acid in a suitable solvent under
standard conditions well known in the art. The formation of such salts is well known and
appreciated in the art. See, for example, 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).
The compounds of the present invention, or salts thereof, may be prepared by a
variety of procedures known to one of ordinary skill in the art, some of which are
illustrated in the schemes, preparations, and examples below. One of ordinary skill in the
art recognizes that the specific synthetic steps for each of the routes described may be
combined in different ways, or in conjunction with steps from different schemes, to
prepare compounds of the invention, or salts thereof. The products of each step in the
schemes below can 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 unless otherwise indicated, are
as previously defined. The reagents and starting materials are readily available to one 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, one of ordinary skill in the art appreciates that the compounds of Formulas Ia,
Ib, Ic, and Id may be prepared by using starting material with the corresponding
stereochemical configuration which can be prepared by one of skill in the art. For
example, the Schemes below utilize starting materials with the configuration
corresponding ultimately to Formula Ia.
Generally, a compound of Formula Ia may be prepared from a compound of
Formula II (Scheme 1). More specifically, a compound of Formula IIa is reductively
alkylated with N-(4-fluoroformylthiazolyl)acetamide in the presence of a suitable
reducing agent such as sodium triacetoxyborohydride in a suitable solvent to provide a
compound of Formula Ia in a suitable solvent, such as ethyl acetate. N-(4-Fluoro
formylthiazolyl)acetamide may be prepared by methods know in the chemical arts as
well as methods provided in the following Preparations and Examples.
A compound of Formula IIa may be prepared from a compound of Formula IIIa
where Pg is a suitable amine protecting group. More specifically, a compound of
Formula IIa where Pg is tert-butyl carboxylate (t-BOC) is reacted with an acid such as
hydrochloric acid or trifluoroacetic acid in a suitable solvent such as dioxane or
dichloromethane to provide a compound of Formula IIa. Suitable amine protecting
groups are known in the chemical arts and include t-BOC and Cbz as well as those
discussed in T. W. Green, P. G. M. Wuts, "Protective Groups in Organic Synthesis"
Wiley-Interscience, New York, 1999.
Scheme 1
deprotection
IIIa
A compound of Formula IIIa where Pg is a suitable amine protecting group may
be prepared from a compound of Formula IVa (Scheme 2). More specifically, a
compound of Formula IVa where Pg is tert-butyl carboxylate is reacted with
3-(chloromethyl)methyl-1,2,4-oxadiazole in the presence of a base such as sodium
tert-butoxide to provide a compound of Formula IIIa. The reaction is conveniently
carried out in a solvent such as acetonitrile or dimethylformamide. A compound of
Formula IVa where Pg is tert-butyl carboxylate may be prepared essentially as described
in A1. More specifically, a compound of Formula Va is reacted with a
reducing agent such as lithium tri(sec-butyl)borohydride in a solvent such as
tetrahydrofuran to provide a compound of Formula IVa where Pg is tert-butyl
carboxylate. A compound of Formula Va where Pg is a suitable amine protecting group
may be prepared by processes known in the chemical arts including those described in
A1.
Scheme 2
IIIa
Preparation 1
Synthesis of tert-butyl N-(4-fluoroformyl-thiazolyl)carbamate.
Cesium fluoride (227 g, 1480 mmol) is added to a solution of tert-butyl N-(4-
chloroformyl-thiazolyl)carbamate (38.8 g, 148 mmol; for preparation of tert-butyl
N-(4-chloroformyl-thiazolyl)carbamate see for example, N. Masuda, et al., Bioorg
Med Chem, 12, 6171-6182 (2004)) in DMSO (776 mL) at room temperature. The
reaction mixture is stirred in a 145 °C heating block with an internal temperature of 133
°C for 48 hours, then the mixture is cooled in an ice-water bath. To the mixture is added
saturated aqueous sodium bicarbonate solution (500 mL), brine (500 mL) and ethyl
acetate (500 mL). The mixture is stirred at room temperature for 10 minutes, then is
filtered through diatomaceous earth, washing with ethyl acetate (500 mL). The filtrate is
transferred to a separating funnel and the layers are separated, then the aqueous layer is
extracted with ethyl acetate (1 L). The combined organics are washed with brine (1 L),
then the brine layer is extracted with ethyl acetate (300 mL). The combined organics are
dried over sodium sulfate, filtered and concentrated to give a residue. The residue is
passed through a pad of silica gel (330 g) eluting with 5% ethyl acetate in
dichloromethane (1.5 L) and the filtrate is concentrated to give a residue (24.2 g).
The residue (32.7 g of combined lots, 133 mmol) is dissolved in isopropanol (303
mL), filtered and then is purified by SFC (Supercritical Fluid Chromatography) using an
IC column (cellulose polysaccharide derivative: tris (3,5-dichlorophenylcarbamate, 30 x
250mm, 5u) with 10%IPA (no additive) at 180 mL/minute with 3 mL injections. The
product-containing fractions are concentrated to give the title compound (16.1 g. MS m/z
247.0 (M+H).
Preparation 2
Synthesis N-(4-fluoroformyl-thiazolyl)acetamide (Method A)
In a jacketed vessel, zinc bromide (91.9 g,408 mmol) is added in one portion to a
mixture of tert-butyl N-(4-fluoroformyl-thiazolyl)carbamate (33.5 g, 136 mmol) and
dichloromethane (503 mL) at room temperature. The reaction mixture is stirred overnight
at an internal temperature of 37 °C, then the jacket temperature is set to −10 °C and
tetrahydrofuran (111 mL) is added dropwise over 15 minutes, maintaining an internal
temperature below 6 °C. The jacket temperature is then set to −30 °C and pyridine (110
mL, 1360 mmol) is added dropwise over 5 minutes, maintaining an internal temperature
below 5 °C. The jacket temperature is set to 0 °C and acetic anhydride (116 mL, 1220
mmol) is added dropwise over 5 minutes. The reaction mixture is stirred overnight at an
internal temperature of 37 °C, then is cooled to room temperature and passed through a
short pad of diatomaceous earth, eluting with tetrahydrofuran (500 mL). The filtrate is
transferred to a flask and the mixture is concentrated to give a residue, which is
concentrated from toluene (50 mL). To the residue is added a solution of citric acid
monohydrate (57.2 g, 272 mmol) in water (400 mL) and 2-methyltetrahydrofuran (400
mL) and the mixture is stirred at 40 °C for 5 minutes, then is passed through a short pad
of diatomaceous earth, eluting with 2-methyltetrahydrofuran (100 mL). The filtrate is
transferred to a separating funnel and the layers are separated. The aqueous layer is
extracted with 2-methyltetrahydrofuran (2 × 250 mL) and the combined organics are
diluted with water (500 mL). To the mixture is added solid sodium bicarbonate
portionwise over 5 minutes with stirring until gas evolution ceases. The mixture is
transferred to a separating funnel and the layers are separated, then the aqueous layer is
extracted with 2-methyltetrahydrofuran (200 mL and 100 mL). The combined organics
are dried over sodium sulfate, filtered and concentrated to give a residue, which is diluted
with 2-methyltetrahydrofuran (100 mL) and the mixture is passed through a short pad of
silica gel (250 g), eluting with 2-methyltetrahydrofuran (2.5 L). The filtrate is
concentrated to give a residue which is suspended in a 1:1 mixture of dichloromethane
and heptane (202 mL). The mixture is stirred at room temperature for 30 minutes and
then filtered. The filtered solid is dried under vacuum at 40 °C for 2 hours to give the title
compound (18.0 g, 70%). MS m/z 189.0 (M+H).
Alternative synthesis of N-(4-fluoroformyl-thiazolyl)acetamide (Method B).
Add dichloromethane (1325 g, 15.6 mol) to 2-aminochlorothiazole
carbaldehyde (100 g, 0.61 mol) and pyridine (194.6 g, 2.46 mol), and cool to 0-5 °C. Add
acetic anhydride (188.4 g, 1.85 mol) dropwise, maintaining the temperature at 0-5 °C.
After addition is complete, adjust the temperature to 20-25 °C and stir for 41 hours.
Concentrate under reduced pressure followed by addition of 35% aqueous HCl (200 mL)
and water (1.5 L), maintaining the temperature at less than 40 °C . Cool to 20-25 °C and
stir for 18 hours. Filter the mixture and wash the collected solid with water. Dry the
solids at 60-65 °C for 24 h to provide N-(4-chloroformylthiazolyl)acetamide (75 g,
0.4 mol).
Under an inert atmosphere, add sulfolane (1000 ml) to the N-(4-chloro
formylthiazolyl)acetamide (50 g, 0.244 mol, prepared directly above),
tetramethylammonium chloride (107.1 g, 0.977 mol), and cesium fluoride (370.6 g, 2.44
mmol). Heat to 130 °C and stir for 23 h. HPLC analysis shows 75% conversion with an
in situ yield of 45% of the title compound.
Alternative synthesis of N-(4-fluoroformyl-thiazolyl)acetamide (Method C).
Add 2-propanol (150 mL) to tetramethylammonium fluoride.tetrahydrate (10.2 g,
109.0 mmol) and concentrate the mixture to 2-3 volumes under vacuum with internal
temperature maintained at 70 °C to remove water. Add 2-propanol (200mL) and
concentrate the mixture to 2-3 volumes under vacuum. Repeat two more times. Add
DMF (200mL) and concentrate to 2-3 volumes under vacuum. Add THF (200 mL) and
concentrate to 2-3 volumes. Repeat two more times. Charge N-(4-chloro
formylthiazolyl)acetamide (1.22 g, 5.96 mmol, prepared above in Method B) and DMF
(12 ml). Heat to 110 °C and stir for 12 h. Cool reaction mixture to 25 °C. Add 2-
methyltetrahydrofuran (40 mL) and water (40 mL). The layers are 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. Add ethyl acetate (20 mL) and
water (5 mL). The layers were separated and the organic layer concentrated to remove
solvent. Add ethyl acetate (2mL) and heptane (2 mL) and filter. The filtered solid is
dried under vacuum at 55 °C for 18 hours to give the title compound as a 93% mixture
with N-(4-chloroformylthiazolyl)acetamide.
Preparation 3
Synthesis of tert-butyl (2S,4S)hydroxymethyl-piperidinecarboxylate.
To a flask is added tert-butyl (2S)methyloxo-piperidinecarboxylate (50 g,
234.44 mmol) and tetrahydrofuran (500 mL). The mixture is cooled to −65 °C under an
atmosphere of nitrogen and lithium tri(sec-butyl)borohydride (304.77 mL, 304.77 mmol;
1 M in tetrahydrofuran) is added dropwise over 45 minutes, maintaining an internal
temperature below −60 °C. The reaction mixture is stirred at room temperature for 1
hour, then is cooled to −30 °C. To the reaction mixture is added a mixture of water
(25.34 mL) and tetrahydrofuran (100.16 mL), maintaining an internal temperature below
−20 °C. An aqueous solution of hydrogen peroxide (118.88 mL, 1.17 mol, 30 wt/wt%) in
water (126.70 mL) is added dropwise over 1 hour, maintaining an internal temperature
below 10 °C. To the mixture is added aqueous hydrogen chloride solution (46.89 mL,
234.44 mmol, 5 M) and methyl t-butyl ether (1.00 L) and the mixture is warmed to room
temperature. The layers are separated and the organic phase is stirred with a solution of
sodium metabisulfite (222.84 g, 1.17 mol) in water (500 mL) for 10 minutes at room
temperature. The layers are separated and the organic phase is dried over magnesium
sulfate and concentrated. The residue is purified by flash chromatography (0-50 %
methyl t-butyl ether /isohexane, silica gel) and the product-containing fractions are
combined and concentrated to give the title compound (40.4 g, 78%). ES/MS (m/e) 238
(M+Na).
Alternative synthesis of tert-butyl (2S,4S)hydroxymethyl-piperidinecarboxylate.
To a glass-lined reactor containing deionized water (460 L), and potassium
dihydrogen phosphate (6.5 kg, 0.41 equiv) at 20 C is charged DMSO (27.4 kg , 1.0 vol)
and D-(+)-glucose monohydrate (28.9 kg, 1.25 equiv). The internal temperature is
adjusted to 30 C, and the pH of the reaction is adjusted to 6.9 by addition of aqueous
sodium hydroxide (8%, 15 L, 0.28 equiv). The reactor is charged with tert-butyl (2S)
methyloxo-piperidinecarboxylate (24.9 kg, 1.0 equiv (99.1%ee)), and the mixture is
agitated at 30 C for 15 min. Ketoreductase (KRED-130, 250 g, 1% w/w), glucose
dehydrogenase (GDH-101, 250 g, 1% w/w), and NADP sodium salt (63 g, 0.25% w/w)
are charged directly to the reaction mixture via an open port. The mixture is maintained
at a temperature of 30 C and pH 7.0 ± 0.2 via addition of 8% aqueous NaHCO . After
stirring for 16.5 h (99.5% conversion), the reaction is charged with Celite™ (12.5 kg, 50
w/w%) and toluene (125 L, 5 vol). After stirring for 30 min at 30 C, the mixture is
transferred to another 2000 L reactor via an in-line GAF-filter (4 sock) over the period of
1 h. The mixture is allowed to stand 30 min without agitation, the layers are separated,
and the aqueous layer is back-extracted with toluene (2 x 125 L). The combined organic
layers are filtered (in-line GAF-filter), and the toluene mixture is washed with aqueous
sodium chloride solution (25%, 125 L, 5 vol) at 25 C. The resulting toluene solution is
azeotropically dried (partial vacuum, internal temp < 60 C) to 0.10 w/w% water, and
cooled to 20 C. The mixture is filtered out of the reactor via a cartridge filter into clean
drums under positive nitrogen pressure. The reaction mixture is then transferred from the
drums into a 500 L glass lined vessel and concentrated under vacuum (< 60 C) to a target
residual volume of 56 L (2.25 vol). n-Heptane (169 kg, 10 vol) is charged at 40 C, and
the mixture is seeded with 25 g of tert-butyl (2S,4S)hydroxymethyl-piperidine
carboxylate. The resulting thick slurry is diluted with additional n-heptane (25 L, 1 vol)
and cooled to 16 C over 4 h. The product is isolated via centrifugation, washing with
n-heptane (25 L per spin; 4 spins necessary), yielding 20.3 kg (81%; >99.9% ee) after
drying for 11 h in a tray dryer at 30 C. ES/MS (m/e) 238 (M+Na).
Preparation 4
Synthesis of tert-butyl (2S,4S)methyl[(5-methyl-1,2,4-oxadiazol
yl)methoxy]piperidinecarboxylate.
3-(Chloromethyl)methyl-1,2,4-oxadiazole (43.5 g, 301 mmol) is added to a
solution of tert-butyl (2S,4S)hydroxymethyl-piperidinecarboxylate (29.5 g, 137
mmol) in acetonitrile (590 mL) at room temperature. The reaction mixture is stirred in an
ice-water bath and sodium tert-butoxide (54.3 g, 548 mmol) is added in portions over 10
minutes, maintaining an internal temperature below 10 °C. The reaction mixture is stirred
in an ice-water bath at an internal temperature of 5 °C for 9 hours, then is warmed slowly
to room temperature and is stirred overnight. The reaction mixture is cooled in an ice-
water bath and saturated aqueous ammonium chloride solution (200 mL) is added over 5
minutes, maintaining an internal temperature below 10 °C during the addition. The
mixture is then diluted with water (100 mL) and warmed to room temperature. The
mixture is extracted with methyl tert-butyl ether (2 × 300 mL) and the combined organics
are washed with brine (300 mL). The combined organics are dried over sodium sulfate,
filtered and concentrated to give a residue. The residue is passed quickly through a pad
of silica gel (300 g) eluting with methyl tert-butyl ether (1 L) and the filtrate is
concentrated to give the title compound (46.5 g, 109%). MS m/z 334.0 (M+Na).
Alternative synthesis of tert-butyl (2S,4S)methyl[(5-methyl-1,2,4-oxadiazol
yl)methoxy]piperidinecarboxylate.
To a solution of tert-butyl (2S,4S)hydroxymethyl-piperidinecarboxylate
(0.25g, 1.16 mmol) and 3-(chloromethyl)methyl-1,2,4-oxadiazole (0.308g, 2.32 mmol)
in N,N-dimethylformamide (3mL) under nitrogen at 0 °C is added portionwise sodium
tert-butoxide (0.35 g, 3.5mmol) over 5 min. The reaction mixture is stirred at rt for 10
min then at 40 °C for 12h. The reaction mixture is cooled to room temperature then
quenched with water (10mL). The layers are separated and the aqueous phase is
extracted with methyl tert-butyl ether (2×10mL). The combined organic extracts are
washed with an aqueous solution of lithium chloride (5%), dried over magnesium sulfate,
filtered and concentrated under reduced pressure to afford the title compound (0.49 g, 0.7
mmol, 81% yield, 60% purity) as a brown oil. MS m/z 334.0 (M+Na).
Preparation 5
Synthesis of 5-methyl[[(2S,4S)methylpiperidyl]oxymethyl]-1,2,4-oxadiazole
hydrochloride.
A flask containing tert-butyl (2S,4S)methyl[(5-methyl-1,2,4-oxadiazol
yl)methoxy]piperidinecarboxylate (4.03 g, 12.9 mmol) is submerged in an ice-water
bath. To this flask is added a 4 M solution of hydrochloric acid in 1,4-dioxane (25.9 mL,
104 mmol) dropwise over 5 minutes with stirring, maintaining an internal temperature
below 20 °C during the addition. The reaction mixture is stirred at room temperature for
1hour, then is concentrated to give the title compound (3.56 g, 92% yield based on 83%
purity measured by H NMR. MS m/z 212.0 (M+H).
Alternative synthesis of 5-methyl[[(2S,4S)methylpiperidyl]oxymethyl]-1,2,4-
oxadiazole hydrochloride.
Add methanol (50 mL) to tert-butyl (2S,4S)methyl[(5-methyl-1,2,4-oxadiazol-
3-yl)methoxy]piperidinecarboxylate (12.9 g, 0.041 mol). The mixture is cooled to 0
°C. A 4M solution of hydrochloric acid in methanol (80 mL) is added dropwise to the
cooled mixture, maintaining an internal temperature below 20 °C. The reaction mixture
is then stirred at room temperature for 18 hours. The mixture is then concentrated to
remove solvent. Acetone (10 mL) is added and the mixture is stirred for 20 min.
Tetrahydrofuran (40 mL) is added and the mixture is stirred for 3 hours. The solid is
collected by filtration under nitrogen and the filtered solid cake is rinsed with
tetrahydrofuran. The filtered solid is then dried under vacuum at 45 °C for 2 hours to
give the title compound as a 90% purity. Recrystallization using acetone can increase
purity of title compound to 95%.
Preparation 6
Synthesis of 5-methyl[[(2S,4S)methylpiperidyl]oxymethyl]-1,2,4-oxadiazole.
To a solution of tert-butyl (2S,4S)methyl[(5-methyl-1,2,4-oxadiazol
yl)methoxy]piperidinecarboxylate (0.49g, 1.6 mmol) in dichloromethane (10mL)
under nitrogen is added trifluoroacetic acid (1.8 mL, 23 mmol). The mixture is stirred at
room temperature for 3h. The mixture is concentrated under reduced pressure to afford a
yellow oil. The residue is dissolved in methanol (5mL) and poured onto a cation
exchange cartridge, eluted with methanol (2×10mL) then a 2 M ammonia solution in
methanol (10mL). The filtrate is concentrated under reduced pressure to give title
compound (0.3g, 1.4 mmol, 91%). MS m/z 212.0 (M+H).
Example 1
Synthesis of N-[4-fluoro[[(2S,4S)methyl[(5-methyl-1,2,4-oxadiazol
yl)methoxy]piperidyl]methyl]thiazolyl]acetamide.
N-(4-Fluoroformyl-thiazolyl)acetamide (28.3 g, 150 mmol) is added to 5-
methyl[[(2S,4S)methylpiperidyl]oxymethyl]-1,2,4-oxadiazole hydrochloride
(48.7 g, 185 mmol, 94% purity) in ethyl acetate (707 mL) at room temperature. The
reaction mixture is stirred at room temperature and N,N-diisopropylethylamine (34.1 mL,
195 mmol) is added dropwise over 1 minute, then sodium triacetoxyborohydride (98.5 g,
451 mmol) is added in one portion. The reaction mixture is stirred in a 31 °C heating
block overnight with an internal temperature of 30 °C, then is cooled in an ice-water bath
to an internal temperature of 5 °C. To the mixture is added 2 M aqueous hydrochloric
acid solution (226 mL) over 15 minutes, maintaining an internal temperature below 10
°C. To the mixture is added water (250 mL) and the mixture is stirred at room
temperature for 5 minutes. The layers are separated and the organic layer is extracted
with a mixture of 2 M aqueous hydrochloric acid solution (28 mL) in water (50 mL). The
first aqueous layer is stirred in an ice-water bath and 50% aqueous sodium hydroxide
solution (25.7 mL) is added dropwise over 10 minutes, maintaining an internal
temperature below 10 °C. The mixture is diluted with saturated aqueous sodium
bicarbonate solution (100 mL), then is stirred at room temperature for 10 minutes and
then is extracted with ethyl acetate (3 × 400 mL). The combined organics are dried over
sodium sulfate, filtered and concentrated to give a residue. The second aqueous layer
from the extraction with aqueous hydrochloric acid is diluted with
2-methyltetrahydrofuran (200 mL) and the mixture is passed through a short pad of
diatomaceous earth. The filtrate is transferred to a separating funnel and the layers are
separated. The aqueous layer is stirred in an ice-water bath and 50% aqueous sodium
hydroxide solution (3.15 mL) is added dropwise over 5 minutes, maintaining an internal
temperature below 10 °C. The mixture is diluted with saturated aqueous sodium
bicarbonate solution (10 mL), then is stirred at room temperature for 5 minutes and then
is extracted with ethyl acetate (3 × 40 mL) and 10% isopropanol in ethyl acetate (100
mL). The combined organics are dried over sodium sulfate, filtered and concentrated to
give a residue, which is combined with the residue from the first part of the workup. The
combined residue is passed through a pad of silica gel (350 g) eluting with ethyl acetate
(3.5 L) and the filtrate is concentrated to give a residue (45.8 g).
The residue (47.5 g of combined lots, 123.9 mmol) is purified by flash
chromatography, eluting with 50-100% ethyl acetate in heptane. The product-containing
fractions are concentrated to residue, which is suspended in a 1:1 mixture of methyl-tert-
butyl ether and heptane (448 mL). The mixture is stirred in a 46 °C heating block for 30
minutes at an internal temperature of 45 °C, then is cooled to room temperature over 2
hours with stirring. The mixture is filtered, washing the solid with a 1:1 mixture of
methyl-tert-butyl ether and heptane (30 mL). The filtered solid is dried under vacuum at
40 °C overnight to give the title compound (28.5 g). MS m/z 384.0 (M+H);
[ α] = +33.4 ° (C=0.26, methanol).
Alternative synthesis of N-[4-fluoro[[(2S,4S)methyl[(5-methyl-1,2,4-oxadiazol-
3-yl)methoxy]piperidyl]methyl]thiazolyl]acetamide.
To a solution of N-(4-fluoroformyl-thiazolyl)acetamide (0.05g, 0.28 mmol) and
-methyl[[(2S,4S)methylpiperidyl]oxymethyl]-1,2,4-oxadiazole (0.04g,
0.19mmol) in dichloromethane (10 mL) under nitrogen are added
N,N-diisopropylethylamine (0.1 mL, 0.57 mmol) and sodium triacetoxyborohydride
(0.12g, 0.57 mmol). The reaction mixture is stirred at room temperature for 12h. The
reaction mixture is poured into a saturated aqueous solution of sodium bicarbonate
(10mL). The layers are separated and the aqueous phase is extracted with
dichloromethane (2×10mL). The combined organic extracts are dried over magnesium
sulfate, filtered and concentrated under reduced pressure to afford an orange oil.
The residue is taken up in methanol (to a total volume of 9.8 ml), filtered and purified
by prep-HPLC (Phenomenex Gemini-NX 10 Micron 50*150mm C-18) (CH CN & Water
with 10 mM ammonium bicarbonate adjusted to pH 9 with ammonium hydroxide, 15 %
to 100% CH CN over 10min at 110ml/min) (1 injection) (271/204 nm) to give the title
compound (0.02g, 0.05 mmol, 28%). MS m/z 384.2 (M+H).
Example 1A
Crystalline N-[4-fluoro[[(2S,4S)methyl[(5-methyl-1,2,4-oxadiazol
yl)methoxy]piperidyl]methyl]thiazolyl]acetamide.
Suspend crude N-[4-fluoro[[(2S,4S)methyl[(5-methyl-1,2,4-oxadiazol
yl)methoxy]piperidyl]methyl]thiazolyl]acetamide (29.9g) in 448 mL of 50% methyl
tert butyl ether in heptane at 46 °C for 30 minutes. Stir the mixture and cool to 19 °C
over two hours before filtering following with a wash of 30 mL of 50% methyl tert butyl
ether in heptane to provide the title compound (28.5g, 95% yield).
X-Ray Powder Diffraction (XRPD) of Example 1A
The XRPD patterns of crystalline solids are obtained on a Bruker D4 Endeavor X-ray
powder diffractometer, equipped with a CuKa source (λ = 1.54060 Å) and a Vantec
detector, operating at 35 kV and 50 mA. The sample is scanned between 4 and 40° in 2θ,
with a step size of 0.0087° in 2θ and a scan rate of 0.5 seconds/step, and with 0.6 mm
divergence, 5.28mm fixed anti-scatter, and 9.5 mm detector slits. The dry powder is
packed on a quartz sample holder and a smooth surface is obtained using a glass slide. It
is well known in the crystallography art that, for any given crystal form, the relative
intensities of the diffraction peaks may vary due to preferred orientation resulting from
factors such as crystal morphology and habit. Where the effects of preferred orientation
are present, peak intensities are altered, but the characteristic peak positions of the
polymorph are unchanged. (see, e.g. The U. S. Pharmacopeia 38 - National Formulary 35
Chapter 941 Characterization of crystalline and partially crystalline solids by X-ray
powder diffraction (XRPD) Official May 1, 2015). Furthermore, it is also well known in
the crystallography art that for any given crystal form the angular peak positions may
vary slightly. For example, peak positions can shift due to a variation in the temperature
or humidity at which a sample is analyzed, sample displacement, or the presence or
absence of an internal standard. In the present case, a peak position variability of ± 0.2 in
2θ will take into account these potential variations without hindering the unequivocal
identification of the indicated crystal form. Confirmation of a crystal form may be made
based on any unique combination of distinguishing peaks (in units of ° 2θ), typically the
more prominent peaks. The crystal form diffraction patterns, collected at ambient
temperature and relative humidity, are adjusted based on NIST 675 standard peaks at 8.85
and 26.77 degrees 2-theta.
A prepared sample of crystalline N-[4-fluoro[[(2S,4S)methyl[(5-methyl-
1,2,4-oxadiazolyl)methoxy]piperidyl]methyl]thiazolyl]acetamide is characterized
by an XRPD pattern using CuKa radiation as having diffraction peaks (2-theta values) as
described in Table 1 below. Specifically the pattern contains a peak at 12.1° in
combination with one or more peaks selected from the group consisting of 15.3°, 21.6°,
22.2°, 22.7°, 23.5°, 24.3°, and 26.8° with a tolerance for the diffraction angles of 0.2
degrees.
Table 1: X-ray powder diffraction peaks of crystalline N-[4-fluoro[[(2S,4S)methyl-
4-[(5-methyl-1,2,4-oxadiazolyl)methoxy]piperidyl]methyl]thiazolyl]acetamide,
Example 1A.
Relative Intensity
Peak Angle (2-Theta °)+/- 0.2°
(% of most intense peak)
1 7.7 9
2 10.1 9
3 12.1 100
4 15.3 50
18.3 11
6 19.3 13
7 21.6 16
8 22.2 16
9 22.7 16
23.5 30
11 24.3 35
12 26.8 27
In vitro human OGA enzyme assay
Generation of OGA proteins
The nucleotide sequence encoding full-length human O-GlcNAc- β-N-
acetylglucosaminidase (NM_012215) is inserted into pFastBac1 (Invitrogen) vector with
an N-terminal poly-histidine (HIS) tag. Baculovirus generation is carried out according
to the Bac-to-Bac Baculovirus Expression system (Invitrogen) protocol. Sf9 cells are
infected at 1.5 x 10 cells/mL using 10 mL of P1 virus per Liter of culture and incubated
at 28 C for 48hrs. Cells are spun down, rinsed with PBS and the pellets stored at -80 C.
The above OGA protein (His-OGA) is purified as follows: 4 L of cells are lysed in 200
mL of buffer containing 50 mM Tris, pH 8.0, 300 mM NaCl, 10% glycerol, 10 mM
Imidazol, 1 mM Dithiothreitol (DTT), 0.1% Triton X-100, 4 tablets of protease
inhibitors (complete EDTA-Free, Roche) for 45 min at 4 C. This cell lysate is then spun
for 40 min at 16500 rpm at 4 C, and supernatant incubated with 6 mL of Ni-NTA resin
(nickel-nitrilotriacetic acid) for 2 hours at 4
Resin is then packed onto column and washed with 50 mM Tris, pH 8.0, 300 mM
X-100, 1 mM DTT, followed by
NaCl, 10% glycerol, 10 mM Imidazole, 0.1% Triton
50 mM Tris, pH 8.0, 150 mM NaCl, 10 mM Imidazol, 10% glycerol, 1 mM DTT. The
proteins are eluted with 50 mM Tris, pH 8.0, 150 mM NaCl, 300 mM Imidazole, 10%
glycerol, 1 mM DTT. Pooled His-OGA containing fractions are concentrated to 6 ml and
The protein is eluted with 50 mM Tris, pH 8.0, 150 mM
loaded onto Superdex75 (16/60).
NaCl, 10% glycerol, 2 mM DTT. Fractions containing His-OGA are pooled and protein
concentration measured with BCA (Bradford Colorimetric Assay).
OGA enzyme assay
The OGA enzyme catalyses the removal of O-GlcNAc from nucleocytoplasmic
proteins. To measure this activity Fluorescein di-N-acetyl-β-N-acetyl-D-glucosaminide
(FD-GlcNAc, Kim, Eun Ju; Kang, Dae Ook; Love, Dona C.; Hanover, John A.
Carbohydrate Research (2006), 341(8), 971-982) is used as a substrate at a final
concentration of 10 μM (in the 96 well assay format) or 6.7 μM (in the 384 well assay
format). This fluorogenic substrate becomes fluorescent upon cleavage by OGA, so that
the enzyme activity can be measured by the increase in fluorescence detected at 535 nm
(excitation at 485nm).
The assay buffer is prepared to give a final concentration of 50 mM
H NaPO -HNa PO , 0.01% bovine serum albumin and 0.01% Triton X-100 in water, at
2 3 2 3
pH 7. The final enzyme concentration is 3 nM (in the 96 well assay format) or 3.24 nM
(in the 384 well assay format). Both assay formats yield essentially equivalent results.
Compounds to be tested are diluted in pure dimethyl sulfoxide (DMSO) using ten
point concentration response curves. Maximal compound concentration in the reaction
mixture is 30 µM. Compounds at the appropriate concentration are pre-incubated with
OGA enzyme for 30 minutes before the reaction is started by the addition of substrate.
Reactions are allowed to proceed for 60 minutes at room temperature. Then, without
stopping the reaction, fluorescence is read. IC values are 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 of 2.36 nM ± 0.786 (n=8). This data demonstrates that the compound of
Example 1 inhibits OGA enzyme activity in vitro.
Whole cell assay for measuring the inhibition of OGA enzyme activity
Cell Plating:
Utilizing standard conditions known in the art, TRex-293 cells modified for
inducible expression of the P301S-1N4R form of the microtubule associated protein tau
are generated and maintained in growth media, consisting of DMEM High Glucose
(Sigma# D5796), supplemented with 10 % Tetracyclin-free Fetal Bovine Serum (FBS,
Sigma F2442), 20 mM HEPES, 5 µg/mL Blasticidin (Life Technologies# A11139-03)
and 200 µg/mL Zeocin (Life Technologies# R250-01). For the experiments, cells are
plated in growth media at 10,000-14,000 cells per well in a Corning Biocoat (356663)
384 well plate coated with poly-D-Lysine, and incubated 20-24 h in a cell incubator at
37ºC/5% CO . Experiments are performed without inducing Tau expression.
Compound treatment:
Compounds to be tested are serially diluted 1/3 in pure DMSO using ten point
concentration response curves and further diluted in growth media. 20-24 h after plating,
cells are treated with test compound in growth media; maximal compound concentration
is 15 µM (0.15% DMSO). The maximum inhibition is defined by replicate measurements
of 15 uM Thiamet G and the minimum inhibition is defined by replicate measurements of
0.15% DMSO treatment. The cells are returned to the incubator at 37ºC/5% CO for 20-
24 hours. Compounds are tested in duplicates within each plate.
Immunostaining:
After 20-24 hours of compound treatment, the media is removed from the assay
plate and 25 μL of 3.7 % Formaldehyde solution (Sigma # F1635) in DPBS (Sigma
#D8537) are added to each well and incubated for 30 minutes. The cells are then washed
once with DPBS and then permeabilized with 0.1% Triton X-100 (Sigma# T9284).
After 30 minutes, cells are washed twice with DPBS and then blocking solution(1%
X-100) is added to each well and incubated for 60 minutes.
BSA/DPBS/0.1% Triton
The blocking solution is removed and a 0.40-0.33 μg/mL solution of O-GlcNAc Protein
antibody (RL2 clone, Thermo, MA1072) in blocking solution is added to the cells and
allowed to sit overnight at 2-8 ̊C. The next day, the cells are washed twice with DPBS
and the secondary antibody, Alexa Fluor 488 goat anti-mouse IgG (Life Technologies #
A11001) at 2 ug/mL in DPBS is added to each well and allowed to sit at room
temperature for 90 min. The secondary antibody is removed, cells washed twice with
DPBS and a solution of DAPI (Sigma #D9564) and RNase (Sigma, R6513) in DPBS at a
concentration of 1 and 50 ug/mL, respectively, is added to each well. The plate is sealed,
incubated for one hour and analyzed on an Acumen eX3 hci (TTP Labtech). All the
incubations and washing steps described above are done at room temperature, except for
the primary antibody.
Analysis and Results:
The plates are analyzed on an Acumen eX3 instrument using a 488 and 405 nm
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 (RL2 clone) and
the FL1 filter is the signal corresponding to the cell nuclei (DAPI). The ratio Total
FL2/Total FL1 (Total fluorescence of each well without object or population selection) is
used for data analysis. The data is normalized to a maximum inhibition as referenced by
a 15 µM treatment of Thiamet G and a minimum inhibition as achieved by a 0.15%
DMSO treatment. The data is fitted with a non-linear curve fitting application (4-
parameters logistic equation) and IC values are calculated and reported.
The compound of Example 1 was tested essentially as described above and
exhibited an IC of 21.9 nM ± 7.3 (n=5). This data demonstrates that the compound of
Example 1 inhibits OGA enzyme activity in a cellular assay.
Claims (16)
1. A compound of the formula: 5 or a pharmaceutically acceptable salt thereof.
2. The compound according to claim 1 wherein the methyl at position 2 is in the cis configuration relative to the oxygen at position 4 on the piperidine ring: or a pharmaceutically acceptable salt thereof. 10
3. The compound according to either claim 1 or claim 2 wherein the compound is N-[4-fluoro[[(2S,4S)methyl[(5-methyl-1,2,4-oxadiazol yl)methoxy]piperidyl]methyl]thiazolyl]acetamide, or a pharmaceutically acceptable salt thereof.
4. The compound according to claim 3 which is N-[4-fluoro[[(2S,4S) 15 methyl[(5-methyl-1,2,4-oxadiazolyl)methoxy] piperidyl]methyl]thiazolyl]acetamide.
5. The compound according to claim 4 wherein the compound is crystalline.
6. The compound according to claim 5 which is characterized by a peak in the X-ray powder diffraction spectrum, at diffraction angle 2-theta of 12.1° in 20 combination with one or more peaks selected from the group consisting of 15.3°, 21.6°, 22.2°, 22.7°, 23.5°, 24.3°, and 26.8°, with a tolerance for the diffraction angles of 0.2 degrees.
7. The use of a compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of Alzheimer’s disease.
8. The use of a compound of any one of claims 1-6, or a pharmaceutically 5 acceptable salt thereof, for the manufacture of a medicament for preventing the progression of mild cognitive impairment to Alzheimer’s disease.
9. The use of a compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of progressive supranuclear palsy.
10. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1-6 for use in therapy.
11. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1-6 for use in the treatment of Alzheimer’s disease.
12. A compound or pharmaceutically acceptable salt thereof according to any one 15 of claims 1-6 for use in preventing the progression of mild cognitive impairment to Alzheimer’s disease.
13. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1-6 for use in treating progressive supranuclear palsy.
14. A pharmaceutical composition, comprising a compound or a pharmaceutically 20 acceptable salt thereof according to any one of claims 1-6 with one or more pharmaceutically acceptable carriers, diluents, or excipients.
15. A process for preparing a pharmaceutical composition, comprising admixing a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-6 with one or more pharmaceutically acceptable carriers, diluents, 25 or excipients.
16. A compound or pharmaceutically acceptable salt thereof according to any one of claims 1-6 and 10-13 substantially as herein described with reference to any example thereof.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201762451137P | 2017-01-27 | 2017-01-27 | |
US62/451,137 | 2017-01-27 | ||
PCT/US2018/014331 WO2018140299A1 (en) | 2017-01-27 | 2018-01-19 | N-[4-fluoro-5-[[(2s,4s)-2-methyl-4-[(5-methyl-1,2,4-oxadiazol-3-yl)methoxy]-1-piperidyl]methyl]thiazol-2-yl]acetamide as oga inhibitor |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ754849A NZ754849A (en) | 2021-10-29 |
NZ754849B2 true NZ754849B2 (en) | 2022-02-01 |
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