SELECTIVE BACE1 INHIBITORS
The present invention relates to certain novel selective BACE1 inhibitors, to
pharmaceutical compositions comprising the compounds, to methods of using the
compounds to treat physiological disorders, and to intermediates and processes useful in
the synthesis of the compounds.
The present invention is in the field of treatment of Alzheimer's disease and other
diseases and disorders involving amyloid (Abeta) peptide, a neurotoxic and highly
aggregatory peptide segment of the amyloid precursor protein (APP) 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 rather than halting, slowing, or reversing the
disease, there is a significant unmet need in the treatment of Alzheimer's disease.
Alzheimer's disease is characterized by the generation, aggregation, and
deposition of Abeta in the brain. Complete or partial inhibition of -secretase (-site
amyloid precursor protein-cleaving enzyme; BACE) has been shown to have a significant
effect on plaque-related and plaque-dependent pathologies in mouse models suggesting
that even small reductions in Abeta peptide levels might result in a long-term significant
reduction in plaque burden and synaptic deficits, thus providing significant therapeutic
benefits, particularly in the treatment of Alzheimer's disease. In addition, two homologs
of BACE have been identified which are referred to as BACEl and BACE2, and it is
believed that BACEl is the most clinically important to development of Alzheimer's
disease. BACEl is mainly expressed in the neurons while BACE2 has been shown to be
expressed primarily in the periphery. (See D. Oehlrtch, Bioorg. Med. Chem. Lett., 24,
2033-2045 (2014)) In addition, BACE2 may be important to pigmentation as it has been
identified as playing a role in the processing of pigment cell-specific melanocyte protein
(See L. Rochin, ei al, Proc. Natl. Acad. Sci. USA, 110(26), 10658-10663 (2013)). BACE
inhibitors with central nervous system (CNS) penetration, particularly inhibitors that are
selective for BACEl over BACE2 are desired to provide treatments for Abeta peptidemediated
disorders, such as Alzheimer's disease.
United States Patent No. 8,158,620 discloses fused aminodihydrothiazine
derivatives which possess BACEl inhibitory activity and are further disclosed as useful
therapeutic agents for a neurodegenerative disease caused by peptide, such as
Alzheimer's type dementia. In addition, United States Patent No. 8,338,407 discloses
certain fused aminodihydrothiazine derivatives having BACEl inhibitory effect useful in
treating certain neurodegenerative diseases, such as Alzheimer-type dementia.
The present invention provides certain novel compounds that are inhibitors o f
BACE In addition, the present invention provides certain novel compounds that are
selective inhibitors of BACEl over BACE2. Furthermore, the present invention provides
certain novel compounds which penetrate the CNS. The present invention also provides
certain novel compounds which have the potential for an improved side-effect profile, for
example through selective inhibition of BACEl over BACE2.
Accordingly, the present invention provides a compound of Formula I :
Formula I
or a pharmaceutically acceptable salt thereof.
In addition, the present invention rovides a compound of Formula la:
or a pharmaceutically acceptable salt thereof.
The present invention further provides a compound of Formula II:
Formula II
wherein R is methyl, ethyl, or cyclopropyl;
or a pharmaceutically acceptable salt thereof.
In addition, the resent invention provides a compound of Formula a :
Formula a
or a pharmaceutically acceptable salt thereof.
The present invention also provides a method of treating Alzheimer's disease in a
patient, comprising administering to a patient in need of such treatment an effective
amount of a compound of Formulas , la, II, or Ha, or a pharmaceutically acceptable salt
thereof.
The present invention further provides a method of treating the progression of
mild cognitive impairment to Alzheimer's disease i a patient, comprising administering
to a patient in need of such treatment an effective amount of a compound of Formulas I,
a, I , or Ila, or a pharmaceutically acceptable salt thereof. The present invention also
provides a method of inhibiting BACE in a patient, comprising administering to a patient
in need of such treatment an effective amount of a compound of Formulas I, a, II, Ila, or
a pharmaceutically acceptable salt thereof. The present invention also provides a method
for inhibiting BACE-mediated cleavage of amyloid precursor protein, comprising
admimstenng to a patient in need of such treatment an effective amount of a compound of
Formulas L a, ii, or I a, or a pharmaceutically acceptable salt thereof. The invention
further provides a method for inhibiting production of Abeta peptide, comprising
administering to a patient in need of such treatment an effective amount of a compound of
Formulas 1, a, II, or Ila, or a pharmaceutically acceptable salt thereof.
Furthermore, this invention provides a compound of Formulas I, a, II, or Ila, or a
pharmaceutically acceptable salt thereof for use in therapy, in particular for the treatment
of Alzheimer's disease or for the treatment of the progression of mild cognitive
impairment to Alzheimer's disease. Even furthermore, this invention provides the use of
a compound of Formulas I, la, II, or I a, or a pharmaceutically acceptable salt thereof, for
the manufacture of a medicament for the treatment of Alzheimer's disease.
The invention further provides a pharmaceutical composition, comprising a
compound of Formulas I, la, II, or I a, 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, a, II, or Ila, or a pharmaceutically acceptable salt
thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients.
This invention also encompasses novel intermediates and processes for the synthesis of
the compounds of Formulas I, la, II, and i a.
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. (Morris, et al. Arch. Neurol, 58, 397-405 (2001); Petersen, el al, Arch.
Neurol, 56, 303-308 (1999)). The term "treating 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.
The term "inhibition of production of Abeta peptide" is taken to mean decreasing
of in vivo levels of Abeta peptide in a patient.
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 the attending diagnostician, as
one skilled in the art, by the use of known techniques and by observing results obtained
under analogous circumstances. n determining the effective amount for a patient, a
number of factors are considered by the attending diagnostician, 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 0.01
to about 20 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 (D.B. Troy, Editor, 21st Edition, Lippincott, Williams & Wilkins,
2006).
The compounds of Formulas I, a, II, or Ha, or pharmaceutically acceptable salts
thereof are particularly useful in the treatment methods of the invention, but certain
groups, substituents, and configurations are preferred. The following paragraphs describe
such preferred groups, substituents, and configurations. It will be understood that these
preferences are applicable both to the treatment methods and to the new compounds of
the invention.
Thus, the compound of Formulas I and II wherein the fused bicyclic ring is in the
CIS configuration, or pharmaceutically acceptable salt thereof, is preferred. For example,
one of ordinary skill in the art will appreciate that the compound of Formula la is in the
C S relative configuration for the centers labeled 1 and 2 as shown in Scheme A below.
In addition, the compound of Formula a is comprised of a core that contains three chiral
centers at the carbon atoms labeled 1, 2, and 3. it is understood by one of skill in the art,
that the numbering system used in Scheme A may not correspond to the numbering
system used for naming compounds herein, and as such, the numbering system used in
Scheme A is for illustrative purposes. The preferred relative configuration for the three
chiral centers of Formula a is shown in Scheme A:
Scheme A
Further compounds of the present invention are:
N-[3-[(4aS,7aS)-2-arnmo-5-(l,l-difluoroethyl)-4,4a,5
d] [ ,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-( 1,2,4-triazol-l -yl)pyrazine-2-carboxamide;
N-[3-[(4aSR,5SR,7aSR)-2-amino-5-(l,l-difluoroethyl)-4,4a,5,7-
tetrahydrofuro [3,4-d] [1,3]thiazin-7a-yi] -4-fluoro-phenyl] -5-(1,2,4-triazol- 1-yl)pyrazine-
2-carboxamide, and pharmaceutically acceptable salts thereof.
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
especially preferred:
N~[3~[(4aS,5S,7aS)-2-amino~5~(l,l-dif]uoroethyl)-4,4a,5,7-te1xahydro&ro[3,4-
d] [ ,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(l ,2,4-triazol- 1-yl)pyrazine-2-carboxamide, and
the pharmaceutically acceptable salts thereof; and
N-[3-[(4aS,5S,7aS)-2-amino-5 -(1,1-di fluoroethyl)-4,4a,5,7-tetrahydrofuro [3,4-
d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(l ,2,4-triazol- 1-yl)pyrazine-2-carboxamide
hydrate.
In addition, N-[3-[(4aS,5 S,7aS)-2-amino-5-(1,1-di fluoroethyl)-4,4a,5 ,7-
tetrahydrofuro [3,4-d] [ ,3]thiazin-7a-yl] -4-fluoro-phenyl] -5-( ,2,4-triazol-l -yl)pyrazine-
2-carboxamide is particularly preferred.
Furthermore, N-[3-[(4aS,5S,7aS)-2-amino-5-(l,l-difluoroethyl)-4,4a,5,7-
tetrahydrofuro [3,4-d] [1,3]thiazin-7a-yl] -4-fluoro-pheny ] -5-(1,2,4-triazol- 1-yl)pyrazine-
2-carboxamide malonate; and
N-[3-[(4aS,5S,7aS)-2-amrao-5-(l,l-difluoroetiiy])-4,4a,5,74elTahydrofuro[3,4-
d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(l ,2,4-triazol- 1-yl)pyrazine-2-carboxamide 4-
methylbenzenesuifonate are especially preferred.
One of ordinar skill in the art will appreciate that compounds of the invention
can exist in tautomeric forms, as depicted below in Scheme B. When any reference in
this application to one of the specific tautomers of the compounds of the invention is
given, it is understood to encompass both tautomeric forms and all mixtures thereof.
Scheme B
Additionally, certain intermediates described in the following preparations may
contain one or more nitrogen or oxygen protecting groups. It is understood that
protecting groups may be varied as appreciated by one of skill in the art depending on the
particular- reaction conditions and the particular- transformations to be performed. The
protection and deprotection conditions are well known to the skilled artisan and are
described in the literature (See for example "Greene 's Protective Groups in Organic
Synthesis", Fourth Edition, by Peter G.M Wuts and Theodora W. Greene, John Wiley
and Sons, Inc. 2007).
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, el ah, "Enantiomers, Racemates, and
Resolutions", John Wiley and Sons, Inc., 8 and E.L. E ie and S.H. Wilen,"
Stereochemistry of Organic Compounds", Wiley-Interscience, 94).
A pharmaceutically acceptable salt of the compounds of the invention, such as a
hydrochloride salt, can be formed, for example, by reaction of an appropriate free base of
a compound of the invention, an appropriate pharmaceutically acceptable acid such as
hydrochloric acid in a suitable solvent such as diethyl ether under standard conditions
well known in the art. Additionally, the fonnation of such salts can occur simultaneously
upon deproteetion of a nitrogen protecting group. The formation of such salts is well
known and appreciated in the art. See, for example, Gould, P.L., "Salt selection for basic
drags," International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R.J., etal.
"Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,"
Organic Process Research and Development, 4 : 427-435 (2000); and Berge, S.M., et ah,
"Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66: 1-19, (1977).
Certain abbreviations are defined as follows: "APP" refers to amyloid precursor
protein; "BSA" refers to Bovine Serum Albumin; "CD!" refers to , -
carbonyldiimidazole; "cDNA" refers to complementary deoxyribonucleic acid; "DCC"
refers to 1,3-dicyclohexylcarbodiimide; "Deoxo-Fluor® " refers to bis(2-
methoxyethyl)aminosulfur trifluoride; "DIG" refers to 1,3-diisopropylcarbodiimide;
"DMAP" refers to 4-dimethylaminopyridine; "DMEM" refers to Dulbecco's Modified
Eagle's Medium; "DMSO" refers to dimethyl sulfoxide; "EBSS" refers to Earle's
Balances Salt Solution; "EDCI" refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride; "ELISA" refers to enzyme-linked immunosorbent assay; "F12" refers to
Ham's F12 medium; "FBS" refers to Fetal Bovine Serum; "Fc" refers to fragment
crystal lizable; "FLUOLEAD™" refers to 4-tert-butyl-2,6-dimethylphenylsu3fur
trifluoride; "FRET" refers to fluorescence resonance energy transfer; "HATU" refers to
(dimethylamino)-N,N-dime^
hexafluorophosphate; "HBTU" refers to (lH-benzotriazo3-l-y3oxy)(dimethylamino)-N,Ndimethylmethaniminium
hexafluorophosphate; "HEK" refers to human embryonic
kidney; "HF-pyridine" refers to hydrogen fluoride pyridine or Olah's reagent or
poly(pyridine fluoride); "HOAt" refers to l-hydroxy -7-azabenzotriazole; "HOBT" refers
to 1-hydroxylbenzotriazole hydrate; "IC50" refers to the concentration of an agent that
produces 50% of the maximal inhibitory response possible for that agent; "IgG " refers to
immunoglobulin-like domain Fc-gamma receptor; "MEM" refers to Minimum Essential
Medium; "PBS" refers to phosphate buffered saline; "PDAPP" refers to platelet derived
amyloid precursor protein; "Ph" refers to a phenyl group; "PyBOP" refers to
(benzolriazol-l-yl-oxytripyrrolidinophosphonivirn hexafluorophosphate); "PyBrOP"
refers to bromo-tris-pyrrolidino phosphoniumhexafluorophosphate; "RFU" refers to
relative fluorescence unit; "RT-PCR" refers to reverse transcription polymerase chain
reaction; "SDS-PAGE" refers to sodium dodecy] sulfate polyacrylamide gel
electrophoresis; "SCX" refers to strong cation exchange; "SFC" refers to super critical
chromatography;" T3P®" refers to propylphosphonic anhydride; "THF" refers to
tetrahydrofuran; "TEMPO" refers to (2,2,6,6-tetramethyl-piperidin- 1-yl)oxyl; "TMEM"
refers to transmembrane protein; "trityl" refers to a group of the formula "(Ph ;
"XtalFluor-E® or DAST difluorosulfinium salt" refers to
(diethylamino)difluorosulfonium tetrafluoroborate or N,N -diethy 1-5,5-
difluorosulfiliminium tetrafluoroborate; and "XtalFluor-M® or morpho-DAST
difluorosulfinium salt" refers to tetrafluoroborate or
difluoro-4-mo holinylsulfonium tetrafluoroborate.
It is understood by one of ordinary skill in the art that the terms "tosylate",
"toluenesulfonic acid", "p-toluenesulfonic acid", and "4-methylbenzene sulfonic acid"
refer to the compound of the following structure;
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 ait, 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. The following schemes, preparations, and examples further
illustrate the invention.
In Scheme I , step A, trimethylsulfonium iodide is treated with an organic base
such as n-butyllithium at a temperature of about -50 °C in a solvent such as THF. A
protected oxymethyl oxirane, protected with a suitable protecting group, such as a trityl
group, is then added to the basic solution at -10 °C and allowed to stir for about 2 hours to
give the protected product of Scheme 1, Step A. "PG" is a protecting group developed
for the amino group or oxygen group such as carbamates, amides, or ethers. Such
protecting groups are well known and appreciated in the art. The protected product of
Step A is reacted with an a-haloester such as tert-butoxy bromoacetate using tetra-Nbutylammonium
sulfate or other quaternary ammonium salt phase transfer catalysts in a
solvent such as toluene and an aqueous inorganic base such as sodium hydroxide at about
room temperature to give the compound of Scheme 1, Step B. S ch alkylations are well
know in the art. Alternatively a base such as 60% sodium hydride in oil with solvents
such as N,N-dimethylformamide or THF and a temperature range of 0 to 100 °C can be
used to give the protected product of Step B. The r t-butoxy carbonyl acetate is
converted to an oxime over a 2-step procedure. A reducing agent such as
isobutvlaluminum hydride in hexanes is added dropwise at a temperature of about -70 °C
followed by the dropwise addition of an aqueous acid such as hydrochloric acid at a
temperature of about -60 °C. The work-up is accomplished with an organic extraction to
give the intermediate material. This material is dissolved in an organic solvent such as
dichloromethane and treated with sodium acetate followed by hydroxylamine
hydrochloride to give the oxime product of Step C. The oxime product of Scheme 1, Step
C can be converted to the bicyclic 4,5-dihydroisoxazole product of Step D in a 3+2
cyclization by several methods such as using an aqueous solution of sodium hypochlorite
or an alternative oxidant such as N-chlorosuccinimide and in a solvent such as tert-butyl
methyl ether, toluene, dichloromethane, or xylene at a temperature of about 10-15 °C or
with heating. The 2-fluoro, 5-bromo phenyl group ca be added to the dihydroisoxazole
by generating the organometallic reagent. The organometallic reagent can be generated
from 4-bromo- 1-fluoro-2-iodo-benzene using halogen-metal exchange with reagents such
as n-butyllithium or isopropylmagnesium chloride lithium chloride complex and dropwise
addition at a temperature range of about -78 °C to 15 °C in a solvent such as THF. A
Lewis acid such as boron trifluoride diethyl etherate is then added to give the product of
Scheme 1, Step E. The resulting bicyclic tetrahydroisoxazole can be treated with zinc in
acetic acid to form the ring opened product of Scheme 1, Step F. A alternate method to
open the isoxazole ring uses Raney Nickel in a polar solvent such as ethanol under
pressure with hydrogenation conditions. The product of Step F can then be reacted with
benzoyl isothiocyanate in a solvent such as dichloromethane or THF at a temperature of
about 5 °C to room temperature to give the thiourea compound of Step G. The thiazine
ring can be formed using trifluoromethanesuifoiiic anhydride and an organic base such as
pyridine in a solvent such as dichloromethane at a temperature of about -20 °C to give the
product of Step H. The hydroxymethyl protecting group suc as a trityl group can be
removed in Scheme 1, Step I using methods well known in the art such as formic acid at
room temperature or with />-toluenesulfonic acid monohydrate in solvents such as
dichloromethane and methanol to give the compound of Step Ϊ The hydroxy methyl ca
be oxidized to the carboxylic acid using oxidizing agents suc as 2-iodoxybenzoic acid
(1BX) at temperatures of 0-22 °C in a solvent such as DMSO or addition of
(diacetoxyiodo)benzene portionwise or all at once in a solvent such as acetonitrile or
acetonitrile and water with stirring at a temperature of about 5-25 °C to give the
compound of Scheme 1, Step J. TEMPO can also be used as a catalyst in the oxidation.
The Weinreb amide is prepared in Scheme 1, Step from the acid product of Step J with
the addition of ,-dimethylhydroxylamine hydrochloride, an organic base, such as
txiethylamme, and a coupling reagent such as HATU. The mixture is stirred at room
temperature to give the product of Step K. Other coupling agents that could be used
include GDI, carbodiimides such as DCC, DIG, or ED or other uronium or
phosphonium salts of non-nucleophilic anions, such as HBTU, PyBOP, and PyBrOP.
The Weinreb amide is then converted to the ketone using an organometallic reagent such
as a Grignard reagent or an organolithium reagent in Step L in a solvent such as THF.
The appropriate Grignard reagent can be added as a solution in solvents such as ether or
2-methyltetrahydrofuran to the Wemreb amide at a temperature of about -78 °C to 0 °C to
give the ketone of Step L. n Scheme 1, Step M, the R and ketone group of the compound
of Step L can be converted to a difluoro-R group using Deoxo-Fluor® in a solvent such
as dichloromethane at about -78 °C to room temperature. Another alternative procedure
involves pre-mixing the fluorinating reagent such as Deoxo-Fluor® with boron
trifluoride-diethyl etherate followed by the addition of the product of Scheme 1, Step L
and iriethylamine trihydro fluoride to give the product of Scheme 1, Step M.
Alternatively, other fluorinating agents that may be used which are well known in the art
are, diethyiaminosulfur trifluoride (also referred to as "DAST"), XtalFiuor-E® or
Xta2F3uor-M® with an additive such as triethylamine trihydrofluoride or FLUOLEAD™
using an additive such as HF-pyridine. The 5-bromo of the phenyl is converted to the
amine using (lR,2R)-N,N'-dimethyl-l,2-cyclohexanediarnine or trans-
N,N'dimethylcyclohexane-l,2 -diamine in a solvent such as ethanol and adding sodium
azide followed by sodium L-ascorbate and cupric sulfate. The reaction is heated to about
80-100 °C for several hours and then worked up with an extraction using a solvent such
as ethyl acetate. The intermediate is then reduced under hydrogenation conditions using
palladium on carbon such as 5-10% palladium in solvents such as methanol or ethanol
and THF at a pressure of about 40-50 psi of hydrogen to give the aniline product of
Scheme 1, Step N.
Scheme 2
Alternatively in Scheme 2, the protected product of Scheme 1, Step A, can be
treated with 4-(2-chloroacetyl)morpholino and a base such as tetrabutyl ammonium
hydrogen sulfate in a solvent such as toluene at a temperature of about 5 °C to give the
product of Scheme 2, Step A. The morpholino group can then serve as a leaving group in
Scheme 2, Step B. For example, the product of Scheme 2, Step A can be treated with the
appropriate Grignard reagent which can be prepared in situ from isopropyl magnesium
chloride lithium chloride complex and 4-bromo-l-fluoro-2-iodobenzene or if the
appropriate Grignard reagent is available, the reagent can be added directly to the product
of Scheme 2, Step A at a temperatixre of about 5 °C to give the product of Scheme 2, Step
B. The carbonyi acetate can be converted to an oxime with hydroxylamine hydrochloride
and sodium acetate with heating to about 50 °C to give the product of Scheme 2, Step C.
The oxime product of Scheme 2, Step C can then be converted to the product of Scheme
2, Step D (the same product as Scheme 1, Step E) using hydroquinone in a solvent such
as toluene and heating to ref x. The amine product of Scheme 2, Step D can be acylated
with acetyl chloride using an organic base such as DMAP and pyridine in a solvent such
as dichloromethane at a temperature of about 0-5 °C to give the product of Scheme 2,
Step E. The product of Scheme 2, Step E can then be converted to the product of Scheme
3, Step A as discussed below.
Scheme 3
In an alternate route, as described in Scheme 3, the isoxazole nitrogen of the
compound of Scheme 1, Step E, is protected with an acetyl group and the protecting
group of the hydroxy methyl is removed in a two-step procedure. For example, the
tetrahydroisoxazole is treated with an organic base such as DMAP and pyridine in a
solvent such as dichloromethane and acetyl chloride is added. The temperature is
maintained below about C and then allowed to stir at about roo temperature. The
reaction is diluted with water and extracted with a solvent such as dichloromethane. The
organic extracts are washed with an aqueous acid such as I N hydrochloric acid and the
aqueous extracted again with a solvent such as dichloromethane followed by an aqueous
wash. The organic solvent can be partially removed and an acid such as formic acid or ptoluenesulfonic
acid monohydrate in solvents such as dichloromethane and methanol can
added to deprotect the hydroxy methyl. The mixture can be stirred at room temperature
or heated to a temperature of about 40 °C until deprotection of the hydroxy is complete to
give the compound of Scheme 3, Step A. The hydroxy methyl product of Scheme 3, Step
A can be oxidized to the carboxylic acid product of Scheme 3, Step B in a manner
analogous to the procedure described in Scheme 1, Step J, and the Weinreb amide can be
further prepared in a manner analogous to the procedure described in Scheme 1, Step
using a coupling agent such as CDI in a portionwise addition or adding at once with a
solvent such as dichloromethane, cooling to -20 °C and stirring for about 1 hour and
adding N,0-dimethylhydroxylamine hydrochloride portionwise or all at once. An organic
base such as triethylamine can also be used to promote the reaction. Further additions of
CDI and ,-dimethylhydroxylamine can be added until complete reaction is observed to
give the Weinreb amide product of Scheme 3, Step C. The ketone of Scheme 3, Step D
can be formed from the Weinreb amide in a manner analogous to the procedure described
in Scheme 1, Step L. The ketone of Step D can be converted to a difluoro-R group i a
manner analogous to the procedure described in Scheme 1, Step M to give the product of
Scheme 3, Step E. The acetyl tetrahydroisoxazole can deprotected under acidic
conditions well known in the art such as using hydrochloric acid and heating to about 100
°C to give the product of Scheme 3, Step F. The bicyclic tetrahydroisoxazole can be
treated with zinc in acetic acid to form the ring opened product of Scheme 3, Step G in a
manner analogous to the procedure described in Scheme I , Step F. The thiazine product
of Scheme 3, Step H can be prepared in a one pot 2 step reaction using benzoyl
isothiocyanate in a manner analogous to the procedure described in Scheme 1, Step G.
The mixture is evaporated to a residue and cyclohexane is added. The mixture is heated
to about 60 °C and methyl rt-buty] ether is added to dissolve the residue. The solution
is filtered and concentrated to dryness. The thiazine ring can then be formed in a manner
analogous to the procedure described in Scheme 1, Step H to give the product of Scheme
3, Step H.
Scheme 4
In Scheme 4, Step A, the aniline product of Scheme 1, Step N can be coupled with
a heteroaromatic carboxvlic acid utilizing coupling conditions well known in the art. One
skilled in the art will recognize that there are a number of methods and reagents for amide
formation resulting from the reaction of carboxylic acids and amines. For example, the
reaction of an appropriate aniline with an appropriate acid in the presence of a coupling
reagent and an amine base such as diisopropylethylamine or triethylamine, will give a
compound of Scheme 4, Step A. Coupling reagents include carbodiimides such as DCC,
DIG, EDCI, and aromatic oximes such as HOBt and HOAt. Additionally, uronium or
phosphonium salts of non-nucleophilic anions such as HBTU, HATU, P OP, and
PyBrOP or a cyclic phosphoric anhydride such as T3P® can be used in place of the more
traditional coupling reagents. Additives such as DMAP may be used to enhance the
reaction. Alternatively, the aniline amine can be acylated using substituted benzoyl
chlorides in the presence of a base such as triethylamine or pyridine. In Scheme 4, Step
B, the protected thiazine amine can then be deprotected with an organic base such as
pyridine and O-methylhydroxylamine hydrochloride in solvents such as THF and ethanoi
and an organic base such as pyridine to provide the compound of Formula II.
Alternatively an inorganic base such as lithium hydroxide in methanol may be
deprotect the thiazine to provide the compound of Formula II.
Scheme 5
Alternatively, i Scheme 5, the bromide product of Scheme 1, Step M is converted
to a protected aniline using trifluoroacetamide, copper iodide, a diamine such as trans,
racemic-N,N'-dimethyl-l,2-cyclohexane diamine, an inorganic base such as potassium
carbonate, and sodium iodide with heating to about 100-130 °C to give the protected
aniline product of Scheme 5, Step A. The protected aniline and thiazine amine can then
be deprotected stepwise. The trifluoroacetamide can be hydrolyzed using a base such as
7 N ammonia in methanol to give an aniline and protected thiazme, the same product of
Scheme 1, Step N. The thiazine can then be deprotected under conditions well known in
the art and described in Scheme 4, Step B using O-methylhydroxylamine hydrochloride
in a solvent such as ethanol and THF with an organic base such as pyridine followed by
heating to about °C or stirring at room temperature followed by concentration and
purification to give the product of Scheme 5, Step B. Alternatively, the order of
deprotection could be reversed with the thiazine deprotected first and the aniline
deprotected last. In Scheme 5, Step C, the aniline product of Step B can then be reacted
with the appropriate carboxylic acid or acid chloride as described in Scheme 4, Step A to
give the products of Formula II
A pharmaceutically acceptable salt of the compounds of the invention, such as a
hydrochloride salt, can be formed, for example, by reaction of an appropriate free base of
Formulas I, la, II, or a and an appropriate pharmaceutically acceptable acid such as
hydrochloric acid, p-toluenesulfonic acid, or malonic acid i a suitable solvent such as
diethyl ether under standard conditions well known in the art. Additionally, the formation
of such salts can occur simultaneously upon deprotection of a nitrogen protecting group.
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,"
The following preparations and examples further illustrate the invention.
Preparation 1
(2S - -Trityloxybut-3-en-2-ol
Scheme , step A : Stir trimethylsulfonium iodide (193.5 g, 948.2 mmol) in THF
(1264 mL) at ambient temperature for 75 minutes. Cool mixture to -50 °C and add nbutyllithium
(2.5 mol/L in hexanes, 379 mL, 948.2 mmol) via cannula, over a period of
30 minutes. Allow the reaction to gradually warm to -30 °C and stir for 60 minutes. Add
(2S)-2-trityloxymethyl oxirane (100 g, 316.1 mmol) portion wise, keeping the
temperature below - 0 °C. After the complete addition, allow the reaction mixture to
warm to room temperature and stir for 2 hours. Pour the reaction into saturated
ammonium chloride, separate the phases, and extract the aqueous phase with ethyl
acetate. Combine the organic layers and dry over magnesium sulfate. Filter and
concentrate under reduced pressure to give a residue. Purify the residue by silica gel
chromatography, eluting with methyl t-butyl ether: hexanes (10-15% gradient), to give
the title compound (56.22 g, 54%). ES/MS m/z 353 (M+Na).
Alternate Preparation 1
(2S)- 1-Trityloxybut-3-en-2-ol
Scheme 2, step A starting material: Add triphenylmethyl chloride (287 g, 947.1
mmol), DMAP (7.71 g, 63.1 mmol) and triethylamine (140 g, 1383.5 mmol) to a solution
of (2S)-but-2-ene-l,2-diol (prepared as in JACS, 1999, 121, 8649) (64.5 g, 631 mmol) in
dichloromethane (850 mL). Stir for 24 hours at 24 °C. Add 1N aqueous citric acid (425
ml,). Separate the layers and concentrate the organic extract under reduced pressure to
dryness. Add methanol (900 mL) and cool to 5 °C for 1 hour. Collect the solids by
filtration and wash with 5 °C methanol (50 mL). Discard the solids and concentrate the
mother liquor under reduced pressure to dryness. Add toluene (800 mL) and concentrate
to a mass of 268 g to obtain the title compound (129 g, 67%) in a 48 wt% solution of
toluene.
Preparation 2
1-Morpholino-2-[(l S)-l -(trityloxymethyl)allyioxy]ethanone
Scheme 2, step A: Add tetrabutyl ammonium hydrogen sulfate (83.2 g, 245.0
mmol) and 4-(2-chloroacetyl)mo hol e (638.50 g, 3902.7 mmol) to a solution of 1-
trityloxybut-3-en-2-o2 ( 832.4 , 2519 mmol) in toluene (5800 mL) that is between 0 and 5
°C. Add sodium hydroxide (1008.0 g, 25202 mmol) in water (1041 mL). Stir for 19
hours between 0 and 5 °C. Add water (2500 mL) and toluene (2500 mL). Separate the
layers and wash the organic extract with water (2 3500 mL) Concentrate the organic
extract under reduced pressure to dryness. Add toluene (2500 mL) to the residue and then
add n-heptane (7500 mL) slowly. Stir for 16 hours. Collect the resulting solids by
filtration and wash with n-heptane (1200 mL). Dry the solid under vacuum to obtain the
title compound (1075.7 g, 98%)
Preparation 3
1-(5-Bromo-2-fluoro-phenyl)-2-[(l S)-l -(trityloxymethyl)ailyloxy]ethanone
Scheme 2, step B: Add a 1.3 M solution of isopropyl magnesium chloride lithium
chloride complex (3079 mL, 2000 mmol) i THF to a solution of 4-bromo-l-fluoro-2-
iodobenze (673.2 g 2237.5 mmol) in toluene (2500 mL) at a rate to maintain the reaction
temperature below 5 °C. Stir for 1 hour. Add the resulting Grignard solution (51 0 mL)
to a solution of l-mo holmo-2-[(lS)-l-(t ityloxymethyl)allyloxy]ethanone (500 g, 1093
mmol) in toluene (5000 mL) at a rate to maintain the reaction temperature below 5 °C.
Stir for 3 hours maintaining the temperature below 5 °C. Add additional prepared
Grignard solution (429 mL) and stir for 1 hour. Add a 1N aqueous citric acid solution
(5000 mL) at a rate to maintain the temperature below 5 °C. Separate the layers and wash
the organic extract with water (5000 mL). Concentrate the solution under reduced
pressure to dryness. Add methanol (2000 mL) to the residue and concentrate to give the
title compound as a residue (793 g, 73.4% potency, 83%).
Preparation 4
1-(5-Bromo-2-fluoro-phenyl)-2-[(1S)- 1-(trityloxymethyl)allyloxy]ethanone oxime
Scheme 2, step C: Add hydroxylamine hydrochloride (98.3 g) to l -(5-bromo-2-
fluoro-phenyl)-2-[(l S)-l -(trityloxymethyl)allyloxy]ethanone (450 g, 707 mmol) and
sodium acetate (174 g) in methanol (3800 mL). Heat the solution to 50 °C for 2 hours.
Cool to 24 °C and concentrate. Add water (1000 mL) and toluene ( 1500 mL) to the
residue. Separate the layers and extract the aqueous phase with toluene (500 mL).
Combine the organic extract and wash with water (2 400 mL). Concentrate the solution
under reduced pressure to give the title compound as a residue (567 g, 6 1.4% potency,
88%).
Preparation 5
rt-Butyl 2~[( 1S)~ 1-(trityloxymethyl)allyloxy]acetate
Scheme I , step B: Add (2S)-l -trityloxybut-3-en-2-ol (74.67 g, 226.0 mmol) to a
solution of tetra-N-butylammonium sulfate (13.26 g, 22.6 mmol) in toluene (376 mL).
Add sodium hydroxide (50% mass) in water ( 11 mL) followed by t rt-butyl-2-
bromoacetate ( 110.20 g, 565.0 mmol). Stir reaction mixture for 18 hours at ambient
temperature. Pour into water, separate the phases, and extract the aqueous phase with
ethyl acetate. Combine the organic layers and dry over magnesium sulfate. Filter the
mixture and concentrate under reduced pressure to give the title compound (77.86 g,
77%). ES/MS m/z 467 (M+Na).
Preparation 6
(lE)-2-[(lS)-l-(Trityloxymethyl)allyloxy]acetaldehyde
H
Scheme 1, step C: Cool a solution of teri-butyi 2-[(lS)-l
(trityloxymethyl)allyloxy]acetate (77.66 g, 174.7 mmol) in dichioromethane (582.2 mL)
to -78 °C. Add a solution of diisobutylaluminum hydride in hexanes ( 1 mol L, 174.7 mL)
dropwise over a period of 35 minutes and maintain the temperature below -70 °C. Stir at
-78 °C for 5 hours. Add hydrochloric acid in water (2 mol/L, 2.1 mL) to the reaction
mixture dropwise, keeping the temperature below -60 °C. Allow the reaction to gradually
warm to ambient temperature and stir for 60 minutes. Separate the organic extract and
wash with saturated sodium bicarbonate. Dry the solution over magnesium sulfate, filter,
and concentrate under reduced pressure to give a residue. Dissolve the residue in
dichioromethane. Add sodium acetate (28.66 g, 349.3 mmol), followed by
hydroxylamine hydrochloride (18.21 g, 262.0 mmol). Stir at ambient temperature for 18
hours. Pour into water, separate the phases, and extract the aqueous phase with
dichioromethane. Combine the organic layers and dry over magnesium sulfate. Filter the
mixture and concentrate under reduced pressure to give the title compound (68.38 g,
10 1 ). ES/MS m/z 386 (M-H).
Preparation 7
(3aR,4S)-4-(Tri1yloxymethyl)-3,3a,4,6-te1iuhydrofuro[3,4-c]isoxazole
Scheme 1, step D: Cool a solution of (lE)-2-[(lS)-l-
(trityloxymethyl)allyloxy]acetaldehyde oxime (55.57 g, 143.4 mmol) in t r t-butyl methyl
ether (717 mL) to 5 °C. Add sodium hypochlorite (5% in water, 5 mL, 430.2 mmol)
dropwise, keeping the temperature below 10 °C. Stir at 10 °C for 30 minutes. Allow the
reaction to warm to 5 °C. Stir at 15 °C for 8 hours. Dilute the reaction mixture with
ethyl acetate and wash with saturated sodium bicarbonate. Separated the phases, wash
the organic phase with a 5% sodium hydrogen sulphite solution and brine. Dry the
solution over magnesium sulfate, filter, and concentrate under reduced pressure to give a
residue. Purify the residue by silica gel chromatography, eluting with 50% methyl tertbutyl
ether/dichloromethane: hexanes (20-27% gradient), to give the title compound
(35.84 g, 65%). ES/MS m z 408 (M- a).
Preparation 8
(3aR,4S,6aR)-6a-(5-Bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-
tetrahydrofuro[3,4-c]isoxazole
Scheme 1, step E: Cool a solution of 4-bromo-l-fluoro-2-iodo-benzene (86.94 g,
288.9 mmol) in THF (144.5 mL) and toluene (1445 mL) to -78 °C. Add n-butyllithium
(2.5 M in hexanes, 120 mL, 288.9 mmol) dropwise, keeping the temperature below -70
°C. Stir for 30 minutes at -78 °C. Add boron trifluoride diethyl etherate (36.5 mL, 288.9
mmol) dropwise, keeping temperature below -70 °C. Stir the solution for 30 minutes at -
78 °C. Add a solution of (3aR,4S)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-
cjisoxazole (55.69 g, 144.5 mmol) in THF (482 mL) dropwise to the reaction, over a
period of 30 minutes, keeping temperature below -65 °C. Stir at -78 °C for 90 minutes.
Rapidly add saturated ammonium chloride, keeping temperature below -60 °C. Pour into
brine, and extract the aqueous phase with ethyl acetate. Combine the organic extract and
dry over magnesium sulfate. Filter and concentrate under reduced pressure to give a
residue. Purify the residue by silica gel chromatography, eluting with 10-15% diethyl
ethenhexanes (0-70% gradient), to give the title compound (36 52 g, 45%). ES/MS
(7 Br/8 1Br) 560/562 [M+H].
Alternate Preparation 8
Scheme 2, step D: Heat a solution of l-(5-bromo-2-fluoro-phenyl)-2-[(lS)-l-
(trityloxymethyl)allyloxy]ethanone oxime (458 g, 502 mmol) and hydroquinone (56.3g
511 mmol) in toluene (4000 m .) to reflux under nitiOgen for 27 hours. Cool the solution
to 24 °C and add aqueous sodium carbonate (800 mL). Separate the layers and extract the
aqueous phase with toluene (300 mL). Combine the organic extract and wash with water
(2 500 mL). Concentrate the solution under reduced pressure to give a residue. Add
isopropyl alcohol (1500 mL) and heat to reflux. Cool to 24 °C and collect the solids by
filtration. Dry the solid under vacuum to obtain the title compound (212 g, 75%).
Preparation 9
l-[(3aR,4S,6aS)-6a-(5-Bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-
tetrahydrof ro[3,4-c]isoxazol- -y ] ethanone
Scheme 2, step E: Add acetyl chloride (35.56 g, 503.9 mmol) to a solution of
(3aR,4S,6aR)-6a-(5-bromo-2-fluoiO-phenyl)-4-(trityioxymethyl)-3,3a,4,6-
tetrahydrofuro[3,4-c]isoxazole (235.3 g, 420 mmol), DMAP (5.13 g, 42.0 mmol), and
pyridine (66.45 g, 840.1 mmol) in dichloromethane (720 mL) under nitrogen, maintaining
internal temperature below 5 °C. Stir for 1 hour and then add water (300 mL) and 1M
sulfuric acid (300 mL). Stir the mixture for 10 minutes and allow the layers to separate.
Collect the organic extract and wash with saturated sodium carbonate (500 mL) and water
(500 mL) Dry the solution over magnesium sulfate. Filter and concentrate under
reduced pressure to give l-[(3aR,4S 6aS)-6a-(5-Bromo-2-fluoro-phenyl)-4-
(trityloxymethyl)-3,3a,4,64etTahydrofuro[3,4-c]isoxazol-l-y3]ethanone (235 g, 93%) as a
grey solid.
Preparation 0
~[(3aR,4S,6aS)~6a-(5-Bromo-2-fjuorophenyl)-4~(hydroxymethyl)tetrahydro~1H,3Hfuro
[3 4-c [1,2]oxazol- 1-y1] ethanone
Scheme 3, step A: In a 20 Ljacketed reactor add acetyl chloride (290 mL, 4075
mmol) to a solution of (3aR,4S,6aR)-6a-(5-bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-
3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole (1996 g, 3384 mmol), DMAP (56.0 g, 458
mmol), pyridine (500 m ,, 6180 mmol) in dichloromethane (10 L) under nitrogen
maintaining internal temperature below 10 °C. After complete addition ( 1 hour) warm to
20 °C and stir overnight. If reaction is incomplete, add acetyl chloride, DMAP, pyridine,
and dichloromethane until complete reaction is observed. Cool the reaction mixture to 0
°C and slowly add water (5 L), stir the reaction mixture at 10 °C for 30 minutes and allow
the layers to separate. Collect the organic extract and wash the aqueous with
dichloromethane ( 1 L). Wash the combined organic extracts with 1N aqueous
hydrochloric acid (2 4 L), extract the aqueous with dichloromethane (2 L). Wash
the combined organic extracts with water (4 L) and remove the solvent under reduced
pressure give total volume of approximately 5 L. Add 90% formic acid (1800 mL) and
stand at ambient temperature for 3 days. Warm to 40 °C for 2 hours then remove the
solvent under reduced pressure. Dilute the residue with methanol (4 L) and slowly add
saturated aqueous sodium carbonate (3 L). Add solid sodium carbonate (375 g) to adjust
the p to 8-9. Stir at 45 °C for 1 hour then cool to ambient temperature. Remove the
solids by filtration, washing with methanol (4 500 mL) then treat with 2 N aqueous
sodium hydroxide (100 mL) and stand at ambient temperature for 1 hour. Remove the
solids by filtration, washing with methanol (2 x 100 mL). Evaporate the solvent under
reduced pressure and partition the residue between ethyl acetate (5 L) and water (2 L).
Extract the aqueous with ethyl acetate (2 L) and wash the combined organic extracts with
brine (2 x 1 L). Remove the solvent under reduced pressure, add methyl feri-butyl ether
(2.5 L) and evaporate to dryness. Add methyl tert-butyl ether (4 L) and stir at 65 °C for 1
hour cool to ambient temperature and collect the solids by filtration, washing with methyl
r t-butyl ether (3 500 mL). Dry under vacuum to a beige solid. Heat this solid in
toluene (7.5 L) to 110 °C until fully dissolved, cool to 18 °C over 1 hour, and stir at this
temperature for 1 hour. Warm to 40 °C and when precipitate forms, cool to 18 °C once
more. Stir for 45 minutes then collect solids by filtration, washing with toluene (2 x 500
mL). Dry the solid under vacuum to obtain the title compound (443.1 g, 36%, 95% purity
by LCMS). Evaporate the filtrate under vacuum to give a residue. Purify the residue by
silica gel flash chromatography, eluting with 20% to 100% ethyl acetate in isohexane.
Slurry the product containing fractions in methyl t rt-butyl ether (2 L) at 60 °C for 30
minutes, cool to ambient temperature, and collect the solids by filtration, washing with
methyl -butyl ether (2 x 200 mL). Dry the solids under vacuum to give the title
compound as a beige crystalline solid (304 g, 24%, 88% purity by LCMS). Evaporate the
filtrate under vacuum to a residue. Purify the residue by silica gel flash chromatography,
eluting with 20% to 00% ethyl acetate in isohexane to give the title compound (57.8 g,
5%, 88% purity by LCMS). ES/MS: m z ( Br/ Br) 360.0/362.0 [M+H].
Alternate Preparation 10
Scheme 3, step A: Add l-[(3aR,4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-
(trityloxymethy3)-3,3a,4,6-tetTahydrofuro[3,4-c]isoxazol-l-yl]ethanone (69 g, 14.5
mmol) to a 15 °C solution ofp-toluenesulfonie acid monohydrate (2.2 g, 11.45 mmol),
dichloromethane (280 mL) and methanol (700 mL). Stir for 18 hours and then remove
the solvent under reduced pressure. Dilute the residue with dichloromethane (350 mL)
and add 1M aqueous sodium carbonate (140 mL) and water (140 mL). Separate the
layers and evaporate the organic layer under reduced pressure. Add toluene (350 mL) to
the residue and heat to reflux for 1 hour. Cool to 10-15 °C at a rate of 10 °C/hour.
Collect the solids by filtration and wash with toluene (70 mL). Dry the solid under
vacuum to obtain the title compound (30 g, 65%) as a grey solid.
Preparation 11
(3aR,4S,6aS)-l-Acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,64etTahydrofuro
c]isoxazole-4-carboxylic acid
Scheme 3, step B: Add water (2 L) to a suspension of l-[(4S,6aS)-6a-(5-bromo-2-
fluoro-phenyl)-4-(hydroxymethyiy^
(804.9 g, 2177 mmoi), TEMPO (40.0 g, 251 mmol) in acetonitrile (4.5 L) in a 20 L
jacketed reactor and cool to an internal temperature of 5 °C. Add (diacetoxyiodo)benzene
(1693 g, 4993.43 mmol) portionwise over 30 minutes. Control the exotherm using
reactor cooling and then hold at 20 °C until LCMS shows complete reaction. Slowly add
a suspension of sodium bisulfite (70 g, 672.68 mmol) in water (300 mL) at ambient
temperature, maintaining the internal temperature below 25 °C. Stir for 30 minutes and
then cool to 5 °C. Add water (2 L), then slowly add 47 wt% aqueous sodium hydroxide
(780 mL) over a period of 1 hour maintaining the internal temperature below 10 °C. Add
ethyl acetate (2 L) and isohexane (5 L), stir vigorously and separate the layers. Extract
the biphasic organic layers with water ( 1 L) and wash the combined aqueous with methyl
tert-butyl ether (2.5L). Cool the aqueous extracts to 5 °C and slowly add 37%
hydrochloric acid ( 1.4 L) over 30 minutes maintaining the internal temperature around 5
°C. Add ethyl acetate (5 L), separate the layers and wash the organic with brine (3 1 L).
Extract the combined aqueous extracts with ethyl acetate (2.5 L), wash the combined
organics with brine ( 1 L), then dr with sodium sulfate, and filter. Dilute the organics
with heptane (2.5 L) and evaporate to dryness under reduced pressure. Add methyl tertbutyl
ether (1.5 L) and heptane (1.5 L) and evaporate to dryness. Add heptane (2.5 L)
and evaporate to dryness twice. Add heptane (500 mL) and methyl t rt-butyl ether (500
mL) and stir at 40 °C for 30 minutes then collect the precipitate by filtration, washing
with heptane/methyl tert-butyl ether (1:1, 1 L) then methyl -buty l ether (3 300 mL)
and air dry to give the title compound as a beige crystalline solid (779 g, 91%). ES/MS:
z ( Br/ Br) 374.0/376.0 [M+H].
[ ] 2 = -19.0 ° (C=1.004, chloroform).
Alternate Preparation 11
Scheme 3, step B: Add water (150 mL) and acetonitrile (150 mL.) to l-[(4S,6aS)-
6a~(5~brQmo-2~fiuoro-pheny3)-4-(hydroxym
1-yljjethanone (30 g, 73.3 mmol), TEMPO (1.14 g, 7.30 mmol) and (diacetoxyiodo)
benzene (51 9 g, 161 mmol). Cool to 15 °C and stir for 2 hours. Slowly add sodium
thiosulfate (21 g) and potassium carbonate (22 g) in water (150 mL) at ambient
temperature. Stir for 1 hour and then add methyl teri-butyl ether (150 mL). Separate the
layers and adjust the p of the aqueous layer to 2-3 with concentrated sulfuric acid. Add
ethyl acetate (150 mL) and separate the layers. Evaporate the organic layer to dryness
under reduced pressure. Add n-heptane (90 mL) and heat to reflux for 1 hour. Cool to 5
°C and then collect the precipitate by filtration, washing with n-heptane (90 mL). Dry
under vacuum to give the title compound as a white solid (27 g, 98%).
Preparation 12
(3aR,4S,6aS)-l-Acety2-6a-(5-bromo-2-fluoropheiiyl)-N-methoxy-N-methyltetrahydro-
1H,3H-furo[3,4-c] [1,2]oxazole-4-carboxamide
Scheme 3, step C: I a 10 L jacketed reactor, cool a solution of (3aR,4S,6aS)-lace1yl-
6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-te1rahydrofuro[3,4-c]isoxazole-4-
carboxylic acid (77 g, 2019 mmol) in dichloromethane (7.0 L) to 0 °C under nitrogen
and add GDI (400 g, 2421 mmol) portionwise over 40 minutes. Cool the reactor jacket to
-20 °C and stir for 1 hour and then add N,0-dimethylhydroxylamine hydrochloride (260.0
g, 2612 mmol) portionwise over about 30 minutes. Stir at -20 °C for 1 hour, at 0 °C for 2
hours, and at 10 °C for 7 hours. Add GDI (175 g, 1058 mmo!) and stir at 10 °C
overnight. Add further GDI (180 g, 1088 mmol) at 10 °C and stirr for 1 hour then add
N,0-dimethylhydroxylamine hydrochloride (140 g, 1407 mmol) and continue stirring at
10 °C. If the reaction is incomplete, further charges of GDI followed by N,0~
dimethylhydroxylamine hydrochloride can be made until complete reaction is observed.
Cool the reaction mixture to 5 °C and wash with 1N aqueous hydrochloric acid (5 L)
then 2 N aqueous hydrochloric acid (5 L). Extract the combined aqueous solution with
dichloromethane ( 1 L), combine the organic extract and wash with water (2,5 L), 1N
aqueous sodium hydroxide (2.5 L), and water (2.5 L), dry over magnesium sulfate, filter,
and evaporate under reduced pressure to give a residue. Add methyl ter t-butyl ether (3 L)
and evaporate under reduced pressure. Add further methyl iert-butyl ether (2 L) and stir
at 50 °C for 1 hour, cool to 25 °C and stir for 30 minutes. Collect the resulting solids by
filtration, wash with methyl ter t-butyl ether (2 500 mL) and dry under vacuum to give
the title compound (760 g, 88%) as a white solid. ES/MS: m z ( Br/ i Br) 417.0/419.0
[M+H].
Alternate Preparation 12
Scheme 3, step C: Cool a solution of (3aR,4S,6aS)-l-acetyl-6a-(5-bromo-2-
fluoro~phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]tsoxazole-4-carboxylic acid (27g, 70.7
mmol) in N,N-dimethylformamide (135 mL) to 0 °C under nitrogen and add CD (14 9 g,
91.9 mmol). Stir for 1 hour and then add ,-dimethylhydroxylamine hydrochloride (9.0
g, 92 mmol) and triethylamine (14.3 g, 141 mmol). Stir at 15 °C for 16 hours. Cool the
reaction mixture to 0 °C and add 0.5 M aqueous sulfuric acid (675 ml,). Stir for 1 hour.
Collect the resulting solids by filtration. Slurry the solids in methyl tert-butyl ether (90
mL) for 1 hour. Collect the solids by filtration, wash with methyl tert-butyl ether (30
mL). Dry under vacuum to give the title compound (23 g, 78%) as a solid.
Preparation 3
-[(3aR,4S,6aS)- 1-Acetyl-6a-(5-bromo-2-fluoro-phenyi)-3,3a,4,6-tetrahydrofiiro[3,4-
c]isoxazol-4-yl]ethanone
Scheme 3, step D: In a 20 L jacketed reactor, cool a solution of (3aR,4S,6aS)-lacetyl-
6a-(5-bromo-2-fluorophenyl)-N-m
c][l,2]oxazole-4-carboxamide (654.0 g, 1536 mmol) in THF (10 L) to -60 °C and add a
3.2 M solution of methylmagnesium bromide in 2-metliyltetraliydrofuran (660 mL, 2110
mmol) dropwise, while maintaining the internal temperature below -40 °C. Stir the
reaction mixture at -40 °C for 30 minutes then cool to -50 °C and add a solution of 1N
aqueous hydrochloric acid (2 L) in THF (2 L) maintaining the mtemal temperature below
-38 °C. Increase the temperature to 10 °C and add ethyl acetate (5 L) and water ( 1 L), stir
and allow internal temperature to reach 5 °C and separate the layers. Extract the aqueous
layer with ethyl acetate ( 1 L) and combine the organic extracts. Wash the organic
extracts with water (2 L) and extract the aqueous layer with ethyl acetate ( 1 L). Combine
the organic extract and wash with brine (3 2 L) then dry over magnesium sulfate, filter,
and evaporate under reduced pressure to a residue. Add cyclohexane (2.5 L), stir at 60 °C
for 1 hour then at 20 °C for 30 minutes, and collect the solid by filtration, washing with
cyclohexane (500 mL). Dry the solid under vacuum to obtain the title compound as a
white solid (565 g, 99%). ES/MS: m/z ( Br/ Br) 372.0/374.0 [M - ] , [ ]D
2 = -58.0 °
(C=1.000, chloroform).
Alternate Preparation 3
Scheme 3, step D: Cool a solution of (3aR,4S,6aS)-l-acetyl-6a-(5-bromo-2-
fluorophenyl)-N-methoxy-N-methyltetrahydiO-lH,3H-furo[3,4-c][l,2]oxazole-4-
carboxamide (4.0g, 9.59 mmol) in THF (60 mL) to -5 °C and add a 3.0 M solution of
methylmagnesium bromide in 2-methyltetrahydrofuran (5.0 mL, 5 mmol) dropwise,
while maintaining the internal temperature between -5 and 0 °C. Stir the reaction mixture
between -5 and 0 °C for 60 minutes then add a solution of saturated ammonium chloride
(20 mL). Add methyl r t-butyl ether (40 mL), allow the internal temperature to reach 5
°C and separate the layers. Evaporate the organic layer under reduced pressure to a
residue. Add n-heptane (50 mL), stir, and collect the solid by filtration. Dry the solid
under vacuum to obtain the title compound as a solid (3.0 g, 77%).
Preparation 4
l-[(3aR,4S,6aS)-6a-(5-Bromo-2-fluoiOphenyl)-4-(Ll-difluoiOethyl)tetrahydiO-lH,3Hfuro
[3,4-c] 1,2]oxazol- 1-yljethanone
Scheme 3, step E: Add l-[(3aR,4S,6aS)-l-acetyl-6a-(5-bromo-2-fluoro-phenyl)-
3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-4-yl]ethanone (5.08 g, 13.6 mmol) in a single
portion to a stirred suspension of XtalFluor-M® (10.02 g, 39.18 mmol) in anhydrous
dichloromethane (100 mL) at 0-5 °C. Stir the mixture for 10 minutes and add
triethylamine trihydrofluoride (4.5 mL, 27 mmol) dropwise over 10 minutes. Stir the
reaction mixture in the ice-bath for 8 hours then warm to ambient temperature and stir
overnight. Add saturated aqueous sodium carbonate (100 m ) and stir for 1 hour.
Separate the layers and extract the aqueous with dichloromethane (2 50 mL). Combine
the organic extracts and wash with saturated aqueous sodium bicarbonate (100 mL), 2 N
aqueous hydrochloric acid (2 00 mL), and brine (100 mL). Evaporate to dryness to a
light brown solid and dissolve in methyl -buty ether (300 mL) at 60 °C. Filter the hot
solution and evaporate the filtrate to give a brown solid (5.3 g, 81%, 82% purity by
LCMS) that is used without further purification. ES/MS: m/z ( Br/ Br) 393.8/395.8
[M+H].
Alternate Preparation 14
Scheme 3, step E: Add XtalFluor-M® (1.21 kg, 4.73 mol) in portions to
solution of l-[(3aR,4S,6aS)-l-acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-
tetrahydrofuro[3,4-c]isoxazol-4-yl]ethanone (565 g, 1.51 mol) in anhydrous
dichloromethane (5 L) at -14 °C. Stir the mixture for 10 minutes and add txiethykmine
trihydrofluoride (550 g, 3 34 mol) dropwise over 20 minutes. Stir the reaction mixture at
-10 °C for approximately 10 hours then warm to ambient temperature and stir overnight.
Add 50% aqueous sodium hydroxide (750 mL) slowly, maintaining the internal
temperature below 10 °C, then add water ( 1.5 L) and saturated aqueous sodium hydrogen
carbonate ( 1 L) and stir for 30 minutes. Separate the layers and extract the aqueous with
dichloromethane ( 1 L). Combine the organic extracts and wash with brine (3 L), 2 N
aqueous hydrochloric acid ( 5 L), and brine (3 L). Evaporate to give a residue and purify
by silica gel chromatography eluting with 50-100% dichloromethane i iso-hexane then
10% methyl t rt-butyl ether in dichloromethane to give the title compound as a white
powder (467 g, 73%, 94% purity by LCMS). ES/MS: m/z ( Br/ iBr) 393.8/395.8
[M+ ]
Preparation 15
(3aR,4S,6aS)-6a-(5-Bromo-2-fluoro-phenyl)~4~(l,l-difluoroethyl)-3,3a,4,6-tetrahydrolH-
furo[3,4-c]isoxazole
Scheme 3, step F: Add 37 wt% aqueous hydrochloric acid (1.3 L, 16 mol) to a
solution of 1-[(3aR,4S,6aS)-6a-(5 -bromo-2-fluorophenyl)-4-( 1,1-
difluoroethyl)tetTahydro-lH,3H-furo[3,4-c][l,2]oxazo3-l-yl]ethanone (570 g, 1.45 mol) in
1,4-dioxane (5 L) in a 10 L jacketed reactor and stir at 100 °C for approximately 3 hours
or until LCMS shows complete reaction. Cool the reaction mixture to 10 °C, dilute with
water ( 1 L) and add a mixture 50 wt% aqueous sodium hydroxide solution (800 mL) and
water ( L) slowly, maintaining the internal temperature below 20 °C. Add ethyl acetate
(2.5 L) and stir vigorously, before separating the layers and washing the organic phase
with brine (2 L), further brine ( 1 L), and water ( 1 L) Dry over magnesium sulfate, filter,
and concentrate to dryness under reduced pressure to give a residue. Add cyclohexane
(2.5 L) and evaporate to dryness then repeat to obtain the title compound as a brown oil
(527 g, 89%, 86% purity by LCMS). ES/MS: m/z ( Br/ Br) 351.8/353.8 [M+H].
Preparation 6
[(2S,3R,4S)-4-Amino-4-(5-bromo-2-fluoropheny
3-yl]methanol
Scheme 3, step G: Add zinc powder (6.0 g, 92 mmol) to a solution of
(3aR,4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-( ,1-difluoroethyl)-3,3a,4,6-tetrahydrolH-
furo[3,4-c]isoxazole (5.06 g, 13.4 mmol) in acetic acid (100 mL) at ambient
temperature and stir overnight. Dilute the mixture with ethyl acetate (200 mL) and water
(300 mL) and stir vigorously while adding sodium carbonate (97 g, 915 mmol). Separate
the layers and wash the organic layer with brine (2 x 200 mL), dry over magnesium
sulfate, filter, and concentrate to give a residue. Purify the residue by silica gel
chromatography eluting with 0% to 100% methyl -butyl ether in isohexane to give the
title compound as a waxy solid (4.67 g, 89%, 90% purity by LCMS). ES/MS: m z
(7 Br/8 1Br) 354.0/356.0 [M+H].
Alternate Preparation 16
Scheme 3, step G: Add zinc powder (200 g, 3.06 mo ) portionwise to a solution
of (3aR,4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-(l,l-difluoroethyl)-3,3a,4,6-tetrahydrolH-
furo[3,4-c]isoxazole (304 g, 75% purity, 647 mmol) in acetic acid (2 L) and water (2
L) at 20 °C then warm to 40 °C and stir overnight. Dilute the mixture water (2 L) and stir
vigorously while adding sodium carbonate (4 kg, 43.4 mol) then adjust to pH 8-9 with
further sodium carbonate. Add ethyl acetate (5 L) and water (2.5 L), stir for 30 minutes
and filter through diatomaceous earth washing with 2:1 acetonitrile/water. Separate the
layers, extract the aqueous with ethyl acetate ( 2.5 L) and wash the combined organic
extracts with brine (2 x 2.5 L), dry over magnesium sulfate, filter, and concentrate to give
a residue. Purify the residue by SFC, column: Chiralpak AD-H (5), 50 250 mm; eluent:
12% ethanoi (0.2% diethylmethylamine in C(¾; flow rate: 340 g/minute at UV 220 n to
give the title compound as a white solid (197.7 g, 84%). [a] 20 = -6.93 ° (0=0.678,
chloroform). ES/MS: m z ( Br/ Br) 354.0/356.0 [M+H].
Preparation 7
[(2S,3R,4S)-4-Armno-4-(5-bromo-2-fluoro-phenyl)-2-^
yljmethanol
Scheme 1, step F: Add (3aR,4S,6aR)-6a-(5-bromo-2-fluoro-phenyl)-4-
(iri1yloxymethy2)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazo2e (31.30 g, 55.9 mol) to acetic
acid (186 mL) to give a suspension. Add zinc (25.6 g, 3 mmol) and stir the reaction
mixture vigorously for 18 hours. Dilute the mixture with toluene and filter through
diatomaceous earth. Concentrate the filtrate under reduced pressure. Solubilize the
residue with ethyl acetate, wash with brine, and saturated sodium bicarbonate. Separate
the phases, dry over magnesium sulfate, filter, and concentrate under reduced pressure to
give the title compound (31.35 g, 99%). ES/MS m/e (7 Br/ Br) 562/564 [M+H].
Preparation 18
N~[[(3S,4R,5S)~3~(5-Bromo~2~fluoro-pheny])-4-(hydroxymethyl)-5-
(1 it oxymethyl)te1 hydrofuran-3-yl]carbamothioyl]benzamide
Scheme 1, step G: Dissolve [(2S,3R,4S)-4-amino-4-(5-bromo-2-fluoro-phenyl)-2-
(tri1yloxyniethy2)terrahydrofuran-3-y2]methanol (31 35 g, 55.73 mmol) in
dichloromethane (557 mL) and cool to 5 °C. Add benzoyl isothiocyanate (9.74 mL,
72.45 mmol). After addition is complete, allow the reaction mixture to warm to room
temperature and stir for 2 hours. Pour into saturated sodium bicarbonate, separate the
phases, and extract the aqueous phase with dichloromethane. Combine the organic
extract and dry over magnesium sulfate. Filter the solution and concentrate under
reduced pressure to give the title compound (42.95 g, 106%). ES/ S m/e ( Br/ iBr)
747/749 [M+Na].
Preparation 9
N-[(4aS 5S,7aS)-7a-(5-Bromo-2-fluorophenyl)-5-(l,l-difluoroethyl)-4a,5,7,7atetrahydro-
4H-ftiro[3,4-d][l,3]tmazin-2-yl]benzamide
Scheme 3, step : Add benzoyl isothiocyanate (1.80 mL, 13.3 mmol,) to a
solution of [(2S,3 R,4S)-4-amino-4-(5-bromo-2-fluorophenyl)-2-( 1,1-
dif]uoroethyl)tetrahydrofuran-3-yl]methanol (4.67 g, 1.9 mmol) in dichloromethane (20
mL) at ambient temperature for 1 hour until LCMS shows reaction is complete.
Evaporate the reaction mixture to a residue under vacuum. Add cvclohexane (50 mL),
warm to 60 °C and add methyl -butyl ether until precipitate is fully dissolved (100
mL). Filter the hot solution, cool to room temperature and slowly evaporate under
reduced pressure until formation of a white precipitate. Remove the solvent under
reduced pressure and dissolve the residue in anhydrous dichloromethane (30 mL), add
pyridine (2.4 mL, 30 mmol), and cool the solution to -25 °C. Add
trifluoromethanesulfonic anhydride (2.2 mL 13 mmol) dropwise over 30 minutes and
allow to warm 0 °C over 1 hour. Wash the reaction mixture with water (25 mL), 2 N
aqueous hydrochloric acid (25 mL), water (25 mL), aqueous saturated sodium
bicarbonate (25 mL), and water (25 mL), dry over magnesium sulfate, filter, and
concentrated to dryness. Purify the residue by silica gel chromatography eluting with 5%
methyl ten-butyl ether i dichloromethane to give the title compound as a light yellow
foam (5.0 g, 76%, 90 % purity by LCMS). ES/MS: m z ( Br/ Br) 499.0/501.0 [M+H]
Alternate Preparation 19
Scheme 3, step : Add benzoyl isothiocyanate (98 mL, 724 9 mmol,) to a
solution of [(2S,3 R,4S)-4-amino-4-(5-bromo-2-fiuorophenyl)-2-( 1,1-
difiuoroethyl)tetrahydrofuran-3-yl]methano3 ( 7.6 g, 546.7 mmol) in dichloromethane
(1.2 L) at 30 °C for 1 hour. Add CD! (101 g, 610.4 mmol) and stir at ambient
temperature for 3 hours. Further charges of GDI can be made to ensure complete
consumption of the thiourea intermediate. Heat to 90 °C for 42 hours and cool the
solution to ambient temperature. Dilute the reaction mixture with ethyl acetate (2 L) and
add 2 N aqueous hydrochloric acid (2 L), stir, add brine ( 1 L) and separate the layers.
Wash the organic layer with 2 N aqueous hydrochloric acid (0.5 L), brine (2 x 1 L) and
aqueous saturated sodium bicarbonate ( 1 L). Dry over magnesium sulfate, filter, and
concentrate to give a residue. Purify the residue by silica gel chromatography eluting
with 0-100% ethyl acetate in iso-hexane to give the title compound as a light yellow solid
(234 g, 83%). ES/MS: m z ( Br/ Br) 499.0/501.0 [M+H].
Preparation 20
N-[(4aS,5S,7aS)-7a-(5-BiOmo-2-fiuoro-pheny3)-5-(trityloxymethyl)-4,4a,
tetrahydrofuro[3,4-d] [1,3]thiazin-2-yl]benzamide
Scheme 1, step H: Dissolve N-[[(3S,4R,5S)-3-(5-bromo-2-fluoro-phenyl)-4-
(hydroxymethyl)-5-(tri1yloxymemyl)tetrahydrofuran-3-yl]carbamo
(42.95 g, 59.18 mmol) in dichloromethane (591 mL) and cool to -20 °C. Add pyridine
(12.0 mL, 148.0 mmol), followed by trifluoromethanesulfonic anhydride (10.97 ml,,
65.10 mmol). Monitor the addition keeping the temperature below -20 °C. Stir the
reaction mixture at -20 °C for 30 minutes. Allow the reaction mixture to warm to room
temperature. Pour into saturated ammonium chloride, separate the phases, and extract the
aqueous phase with dichloromethane. Combine the organic extract and dry over
magnesium sulfate. Filter the solution and concentrate under reduced pressure to give the
title compound (45.24 g, 108%). ES/MS m/e ( Br/ Br) 707/709 [M-i!j.
Preparation 2 1
N-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluoro-phenyl)-5-(hydroxymethyl)-4,4a,
tetrahydro u -d][1,3]thiazin-2-yl]benzamide
Scheme 1, step I : Dissolve N-[(4aS,5S,7aS)-7a-(5-bromo-2-fluoro-phenyl)-5-
(trityloxymethyl)-4,4a,5 ,7-tetrahydrofuro[3,4-d] [1,3]thiazin-2-yi]benzamide (45 .24, 63.93
mmol) in formic acid (160 mL) and stir at ambient temperature for 1 hour. Add water (29
mL) over a period of 5 minutes. Stir for 50 minutes. Concentrate the mixture under
reduced pressure to a residue. Dissolve the residue i methanol (639 mL), add
triethylamine (26.7 mL, 191.8 mmol), and stir overnight at ambient temperature. Pour
into brine, separate the phases, and extract the aqueous phase with chloroform. Combine
the organic extract and dry over magnesium sulfate. Filter and concentrate under reduced
pressure to give a residue. Purify the residue by silica gel chromatography, eluting with
acetone: hexanes (25-38% gradient), to give the title compound (16.04 g, 54%) ES/MS
m/e ( Br/ Br) 465/467 [M+H].
Preparation 22
(4aS,5S,7aS)-2-Benzamido-7a-(5-bromo-2-f!uoro~phenyl)~4,4a,5,7-tetrahydrofuro[3,4-
d] [L3]thiazine-5-carboxylic acid
Scheme 1, step J : Add N~[(4aS,5S,7aS)-7a~(5~bromo-2-fluoro-phenyl)-5-
(hydroxymethyl)-4,4a,5,7-terrahydrofuro[3,4-d][l,3]thiazin-2-yl]beiizamide (16.04 g,
34.47 mmol) to DMSO (172 mL) Add 2-iodoxybenzoic acid (35.56 g, 120.70 mmol)
and stir at ambient temperature for 3 hours. Dilute the reaction mixture with chloroform
(300 mL) and pour into saturated ammonium chloride (400 mL). Separate the organic
phase and dry over magnesium sulfate. Filter the solution and concentrate under reduced
pressure to give a residue. Dissolve the residue in ethyl acetate (400 mL) and wash with
saturated ammonium chloride (2x250 mL). Separate the organic phase, dry over
magnesium sulfate, filter, and concentrate under reduced pressure to give a residue.
Dissolve the residue in a dichloromethane: methanol mixture and add diethyl ether until a
solid precipitates. Collect the solid by filtration and dry under reduced pressure to give
the title compound (5.78 g, 35%). ES/MS m/e (7 Br/ Br) 479/481 [M+H].
Preparation 23
laS,5S,7aS)-2-Benzarmdo-7a-(5-bromo-2-fluoro-phenyl)-N-methoxy-N-methyl-
4,4a,5,7-tetrahydrofuiO[3,4-d][l,3]thiazine-5-carboxamide
Scheme 1, step K: Dissolve (4aS,5S,7aS)-2-benzamido-7a-(5-bromo-2-fluorophenyi)-
4,4a,5 74etrahydrofuro[3,4-d][L3]thiazine-5-carboxylic acid (5.78 g, 12,1 mmol)
in dichloromethane (201 mL) and ,-dimethylhydroxylamine hydrochloride (1.76 g,
18.1 mmol). Add triethylamine (5.29 mL, 36.2 mmol) followed by HATU (7.02 g, 18.1
mmol). Stir at ambient temperature for 3 days. Pour into saturated ammonium chloride,
separate the phases, and extract the aqueous phase with ethyl acetate. Combine the
organic extracts and dry over magnesium sulfate. Filter and concentrate under reduced
pressure to give a residue. Purify the residue by silica gel chromatography, eluting with
ethyl acetate; dichloromethane (0-50% gradient) to give the title compound (4.15 g, 66%).
ES/MS m/e ( Br/ Br) 522/524 [M+H].
Preparation 24
N-[(4aS,5S,7aS)-5-Acetyl-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydrofliro[3 4-
d] 1,3]thiazin-2-yl]benzamide
Scheme 1, step L: Add dropwise to a -78 °C solution of (4aS,5S,7aS)-2-
benzarnido-7a-(5-bromo-2-fluoro-phenyl)-N-methoxy-N-methyl-4,4a,5,7-
terrahydrofuro[3,4-d][l,3]thiazine-5-carboxamide (1.51 g, 2,89 mmol) in THF (57.8 mL)
methylmagnesium bromide (3.0 mol/L in diethyl ether, 4.8 mL, 14.5 mmol). Stir the
reaction at -78 °C for 5 minutes and allow to gradually warm to ambient temperature.
Stir for 30 minutes. Quench the reaction with methanol (4 mL), dilute with saturated
ammonium chloride, and extract with ethyl acetate. Combine the organic extract and dry
over sodium sulfate. Filter and concentrate under reduced pressure to g ve a residue.
Purify the residue by silica gel chromatography, eluting with ethyl acetate: hexanes (0-
100% gradient) to give the title compound (1.28 g, 93%). ES/MS m/e ( Br/ Br) 477/479
[M+Na]
Preparation 24b
N-[(5SJaS)-7a-(5-Bromo-2-fluorophenyl)-5-propanoyl-4a,5,7JA-tetrahydro-4Hfuro[
3,4-d] [ ,3]thiazin-2-yl]benzamide
Scheme 1, step L; Add (4aS,5S,7aS)-2-benzamido-7a-(5-bromo-2-fluoropheiiy3)-
N-methoxy-N-methyl-4,4a,^^
(3.00 g, 5.74 mmol) to THF ( 1 15 mL) under nitrogen and cool to -78 °C. Add a solution
of ethylmagnesium bromide (1.0 mo L) in THF (28.7 mL, 28.7 mmol) to the reaction
dropwise, maintaining the temperature at -78 °C. After 1 hour, add methanol (10 mL) in
one portion and then pour onto saturated ammonium chloride solution. Extract the
mixture with ethyl acetate, dry the organic extracts over magnesium sulphate, filter, and
concentrate in vacuo to give a residue. Purify the residue by silica gel chromatography,
eluting with hexanes/ethyl acetate (100-40% gradient) to give the title compound (2 844
g, 5.788 mmol, 100%). ES/MS m z ( Br/ Br) 491/493/513/515 (M+H a).
Preparation 24c
N-[(5S,7aS)-7a-(5-Bromo-2-fluoiOphenyi)-5-(cyc2opropylcarbonyl)-4a,5,7,7a-tetrahydiO-
4H-furo [3,4-d] [ ,3]thiazin-2-yl]benzamide
Scheme 1, step L: Add dropwise to a -78 °C solution of (4aS,5S,7aS)-2-
benzarnido-7a-(5-bromo-2-fluoro-phenyl)-N-methoxy-N-methyl-4,4a,5,7-
tefrahydrofuro[3,4-d][l,3]thiazine-5-carboxainide (1.51 g, 2 89 mmol) in THF (51.2 mL)
cyclopropylmagnesium bromide (1.0 mol/L in 2-methyltetrahydroturan, 14 mL, 14.4
mmol). Stir the reaction at -78 °C for 5 minutes and allow to gradually warm to ambient
temperature. Stir for 30 minutes. Quench the reaction with methanol (4 mL), dilute with
saturated ammonium chloride, and extract with ethyl acetate. Combine the organic
extract and dry over sodium sulfate. Filter and concentrate under reduced pressure to
give a residue. Purify the residue by silica gel chromatography, eluting with ethyl
acetate: hexanes (0-100% gradient) to give the title compound (1.299 g, 89%). ES/MS
m/z ( Br/ Br) 503/505 [M+Ffj.
Preparation 25
N-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluoiO-phenyl)-5-(l,l-difluoroethyi)-4,4a,5,7-
tetrahydrofuro[3,4-d] [1,3]thiazin-2-yl]benzamide
Scheme 1, step M: Add together dichloromethane (34 mL), Deoxo-Fluor® ( 1.52
mL, 6.88 mmol), and boron trifluoride diethyl etherate (0.89 mL, 6.88 mmol). Stir at
ambient temperature for hours. Add N-[(4aS,5S,7aS)-5-acetyl-7a-(5-bromo-2-fluorophenyl)-
4,4a,5,7-tetrahydrofuro[3,4-d][l,3]thiazm-2-yl]benzamtde (0.821 g, 1.72 mmol)
in one portion, followed by triethylamine tiihydrofluoride (1.13 mL, 6.88 mmol). Stir at
ambient temperature for 18 hours. Pour into saturated ammonium chloride, separate the
phases, and extract the aqueous phase with ethyl acetate. Combine the organic extract
and dry over magnesium sulfate. Filter and concentrate under reduced pressure to give a
residue. Purify the residue by silica gel chromatography, eluting with dichloromethane:
hexanes (80-100 % gradient), to give the title compound (0.552 g, 64%). ES MS m/e
( Br/ Br) 499/501 [M+H].
Preparation 25b
-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluorophenyl)-5-(l,l-difluoropropyl)-4a,5,7,7atetrahydro-
4H-furo[3,4-d] [1,3]thiazin-2-yl]benzamide
Scheme 1, step M: AddN-[(5S,7aS)-7a-(5-bromo-2-fluorophenyl)-5-propanoyl-
4a,5,7,7A-tetrahydro-4I-i-furo[3,4-d][l,3]thiazm-2-yl]benzamide (0.486 g, 0.989 mmol)
to dichloromethane (19.8 mL) under nitrogen at -78 °C, followed by Deoxo-Fluor® (1.75
g, .75 mL, 3.96 mmol). Stir for 30 minutes at -78 °C and then warm to room
temperature overnight. Pour the reaction onto saturated sodium bicarbonate solution and
extract with dichloromethane. Separate organics using a hydrophobic frit and concentrate
in vacuo to give a residue. Purify the residue by silica gel chromatography, elutmg with
hexanes/ethyl acetate (100-50% gradient) to give the crude title compound with an
impurity (0.230 g, 0.448 mmol, 45%) that is used without further purification. ES/MS
ni/z 513/515 (M+H).
Preparation 25c
N-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluoro-phenyl)-5-[cyclopropyl(difluoro)methyl]-
4,4a,5 ,7-tetrahydrofuro[3,4-d] [1,3]thiazin-2-yl]benzamide
Scheme 1, step M: Add Deoxo-Fluor® (1.9 mL, 6.68 mmol) to an ambient
solution of N-[(5S,7aS)-7a-(5-bromo-2-fluorophenyl)-5-(cyclopropy]carbonyl)-4a,5,7,7atetrahydro-
4H-furo[3,4-d][l,3]thiazin-2-yl]benzamide (0.812 g, 1.61 mmol) in
dichloromethane (24 mL). Stir at ambient temperature for 18 hours. Pour into saturated
sodium bicarbonate and extract the aqueous phase with ethyl acetate (3x). Combine the
organic extracts and dry over sodium sulfate. Filter and concentrate under reduced
pressure to give a residue. Purify the residue by silica gel chromatography, eluting with
ethyl acetate; hexanes (5-100 % gradient), to give the title compound (4 mg, 5%).
ES/MS mix ( Br/ Br) 525/527 [M+H].
Preparation 26
N-[(5S,7aS)-5-(l,l-Difluoroethyl)-7a-{2-fluoro-5-[(trifluoroa(x1yl)arnino]phenyl}-
4a,5,7,7a-tetrahydiO-4H-furo[3,4-d] [1,3]thiazin-2-yl]benzamide
Scheme 4, step A : Dissolve N-[(4aS,5S,7aS)-7a-(5-bromo-2-fluorophenyl)-5-
(l,l-difluoroethyl)-4a,5,7,7a4etrahydro-4H-furo[3,4-d ][13]thiazin~2 yl]benzamide (234
g, 454.6 mmol) in 1,4-dioxane (2 L) and add 4 Amolecular sieves (37 g), 2,2,2-
trifluoroacetamide (91 g, 780.9 mmol), finely ground potassium carbonate ( 114 g, 824.9
mmol), sodium iodide (117 g, 780.6 mmol), copper (I) iodide (17.5 g, 91.9 mmol) and
racemic trans-N,N' -dimethyl- 1,2-cyclohexane diamine (20 g, 140.6 mmol) under a
stream of nitrogen. Purge the vessel with 3 vacuum nitrogen switches and heat to 123 °C
for 18 hours. Cool to ambient temperature and filter the solution through diatomaceous
earth, and wash with ethyl acetate. Add saturated aqueous ammonium chloride (2 L) and
vigorously stir for 45 minutes. Separate the layers and wash the organic layer with
saturated aqueous ammonium chloride (3 x 1 L), brine (300 mL), dry over magnesium
sulfate, filter, and evaporate to give a residue. Purify the residue by silica gel
chromatography eluting with 0-100% ethyl acetate in iso-hexane to give the title
compound as a light yellow solid (297.9 g, 95%, 81% purity). ES/MS: m z 532.0 [M+Hj.
Preparation 27
N-[(4aS,5S,7aS)-7a-(5-Arnino-2-fluoro-phenyl)-5-(l,l-difluoroethyl)-4,4a,5,7-
terraliydrofuro[3,4-d] [1,3]thiazin-2-yl]benzamide
Scheme 1, step N: Combine N-[(4aS,5S,7aS)-7a-(5-bromo-2-fluoro-phenyl)-5-
(1,1 -difluoroethyl)-4,4a,5,7-tetrahydrofuro [3,4-d] [1,3] thiazin-2-yl]benzamide (0.372 g,
0.74 mmol) and (lR,2R)-N,N'-dimethyl~l,2-cyclohexanediamtne (0.037 mL, 0.22 mmol)
in ethanol (30 ml). Add sodium azide (0.194 g, 2.98 mmol), followed by sodium Lascorbate
(0.66 M solution, 0.50 ml, 0.33 mmol). Purge the top of the flask with nitrogen
and add cupric sulfate (0.33 M solution, 0.68 ml, 0.22 mmol). Heat the reaction mixture
to 80 °C and stir for 5 hours. Cool the reaction and add cold water. Extract the mixture
with ethyl acetate. Combine the organic extract and dry over sodium sulfate. Filter and
concentrate under reduced pressure to give a residue. Combine the residue with
palladium (10 mass% on carbon, 0.35 g, 0.16 mmol) in ethanol (50 ml) and THF (10 ml).
Purge the mixture with nitrogen and with hydrogen. Stir at ambient temperature under 50
psi of hydrogen for 1 hour. Filter off the catalyst and wash with ethyl acetate.
Concentrate the solution under reduced pressure to give a residue. Purify the residue by
silica gel chromatography, eluting with ethyl acetate: dichloromethane (0-20% gradient),
to give the title compound (0.2184 g, 67%). ES/MS m/z 436 (M+H)
Alternate Preparation 27
Scheme 4, step B : Add 7 N ammonia i methanol (600 mL, 4.2 mol) to a stirred
suspension of N-[(5S,7aS)-5-(l,l-difluoroethy2)-7a-{2-fluoro-5-
[(trifluoroacetyl)amino]phenyl} -4a,5,7,7a-tetrahydro-4H-furo[3,4-d] [ ,3]thiazin-2-
yljbenzamide (250 g, 80% purity, 376.3 mmol) in methanol (200 mL) at room
temperature and stir at ambient temperature for 18 hours. Evaporate to dryness to give
the title compound as a brown gum (190 g, 375.2 mmol, 86% purity). ES/MS: m/z 436.0
[M+H].
Preparation 27b
S 5S,7aS)-7a-(5- Amino-2-fluorophenyl)-5-(l ,1~difluoropropyl)-4a,5 ,7,7a-tetrahydro-
4H- ur -d][1,3]thiazm-2-amine
Scheme 1, ste : Add together N-[(4aS,5S,7aS)-7a-(5-bromo-2-fluorophenyl)-5-
(1,1 -difluoropropyl)-4a,5,7,7a-tetrahydro-4H-ruro [3,4-d] [ ,3]thiazin -2-yl]benzamide
(0.567 g, 1.104 mmol), 1,4-dioxane (4.802 mL) and ethanol ( 1.04 mL), followed by
sodium azide (0.2154 g, 3.313 mmol), trans-,'-dimethyIcyclohexane- ,2-diamine
(0.04760 g, 0.0528 mL, 0.3313 mmol), sodium L-ascorbate 0.66 M (0.74 g, 0.74 mL,
0.4859 mmol) and water (0. 1699 mL). Finally add cupric sulphate 0.33 M (0.74 g, 0.74
mL, 0.2430 mmol) and heat to 100 °C. Stir overnight at 90 °C. Add further cupric
sulfate 0.33 M (0.74 g, 0.74 mL, 0.2430 mmol), sodium L-ascorbate 0.66 M (0.74 g, 0.74
mL, 0.4859 mmol), sodium azide (0.2154 g, 3.313 mmol) and trans-,-
dimethyIcyclohexane- 1,2-diamine (0.04760 g, 0.0528 mL, 0.3313 mmol). Heat the
reaction at 100 °C for 1 hour, and then cool to room temperature. Pour the reaction onto
brine and extract the product with chloroform. Filter mixture through diatomaceous earth
and separate the organics, dry over magnesium sulphate, filter, and concentrate in vacuo.
Transfer the material to a Parr flask with palladium (5 mass%) in Lindlar's catalyst (0.1 13
g, 0.0533 mmol), and add methanol (55.22 mL) under nitrogen. Hydrogenate under 276
kPa hydrogen pressure with vigorous shaking for 3 hours. Add further palladium (5
mass%) in Lindlar's catalyst (0. 3 g, 0.0533 mmol) and hydrogenate under 345 kPa
hydrogen pressure for a further 4 hours. Filter the reaction through diatomaceous earth,
and wash with chloroform. Concentrate the filtrate in vacuo and then add methanol
(55.22 mL) followed by lithium hydroxide hydrate (0.4634 g, 0.182 mL, 11.04 mmol).
8
Heat the reaction mixture to 70 °C for 3 hours and then cool to room temperature. Pour
the reaction onto brine and extract with chloroform. Dilute the organics with methanol,
and pour onto an SCX-2 cartridge. Flush the cartridge with one column volume of
methanol and discard. Then flush the SCX-2 cartridge with one column volume of 7 M
methanolic ammonia and concentrate in vacuo to give the title compound (0.290 g, 0.840
mmol, 76%). ES/MS m/z 346 (M+H).
Preparation 27c
N-[(4aS,5S,7aS)-7a-(5-Arnmo-2-fluoro-phenyl)-5-[cyclopropyl(difluoro)methyl]
4,4a,5,7-tetrahydrofuro[3,4-d] [1,3]thiazin-2-yl]benzamide
Scheme 1, ste : Combine N-[(4aS,5S,7aS)-7a-(5-bromo-2-fluoro-pheny])-5-
[cyclopropyl(difluoro)methyl] -4,4a,5 ,7-tetrahydrofuro[3 ,4-d] [1,3]thiazin-2-yl]benzamide
(3 mg, 0.059 mmol) and (lR,2R)-N N'-dimethyl-l,2-cyclohexanediamine (0.0049 L,
0.030 mmol) in ethanol (3 ml). Add sodium azide (31 mg, 0.47 mmol), followed by
sodium L-ascorbate (0.66 M solution, 0.089ml, 0.059 mmol). Purge the top of the flask
with nitrogen and add cupric sulfate (0.33 M solution, 0.18 ml, 0.059 mmol). Heat the
reaction mixture to 80 °C and stir for 3 hours. Cool the reaction and add cold water.
Extract the mixture with ethyl acetate. Combine the organic extract and dry over sodium
sulfate. Filter and concentrate under reduced pressure to give a residue. Combine the
residue with palladium (10 mass% on carbon, 30 mg, 0.014 mmol) in ethanol (20 ml) and
THF (5 ml). Purge the mixture with nitrogen and with hydrogen. Stir at ambient
temperature under hydrogen at 40 psi for 4 hours. Filter off the catalyst and wash with
ethyl acetate. Concentrate the solution under reduced pressure to give a residue. Purify
the residue by silica gel chromatography, eluting with ethyl acetate: hexanes (0-100%
gradient), to give the title compound (21 mg, 77%). ES/MS m z 462 (M+H).
Preparation 8
aS)-7a-(5-Ammo-2-fluorophenyl)-5-(l,l-difluoroethyl)-4a,5,7,7a-tetrahyd]
4H-furo [ ,4-d] [1,3]thiazm-2-ainine
Scheme 4, step B : Dissolve N-[(4aS,5S,7aS)-7a-(5-ammo-2-fluoro-phenyl)-5-
(1,1 -difluoroethyl)-4,4a,5,7-tetrahydroruro [3,4-d] [1,3]thiazin~2~yl]benzamide (216 4 g,
88% purity, 435.9 mmol) in pyridine (400 mL), ethanol (100 mL) and THF (300 mL).
Add O-methylhydroxylamine hydrochloride (190 g, 2275.0 mmol) and stir at ambient
temperature for 18 hours. Dilute with 2-methyltetrahydrofuran ( 1 L) and wash with water
(2 300 mL). isolate the organic layer and add 35% aqueous ammonium hydroxide (100
mL) to the aqueous. Extract with 2-methyltetrahydrofuran (300 mL) then saturate with
sodium chloride and extract with 2-methyltetrahydrofuran (2 x 300 mL). Combine the
organic extracts, wash with brine (300 mL) and evaporate to a residue. Dissolve in
methanol (200 mL), add 7 N ammonia in methanol (100 mL, 700 mmol) and stir at room
temperature for 8 hours. Further ammonia can be added if any trifluoracetamide
impurity remains. Remove the solvent under reduced pressure and dissolve the residue in
aqueous 2 N aqueous hydrochloric acid (1.5 L). Extract with dichloromethane (6 500
mL), combine the organic layers and remove the solvent under reduced pressure to a total
volume of about 1 L. Wash with 2 N aqueous hydrochloric acid (300 mL) and combine
all aqueous washings. Add 2-methyltetrahydrofuran ( 1 L) and stir vigorously while
adjusting the pH to basic with sodium bicarbonate until no gas evolution is observed.
Separate the layers and e ract the aqueous with 2-methyltetrahydrofuran (2 500 mL).
Dry the combined organic extracts with magnesium sulfate, filter, and evaporate to give a
brown solid. Purify the residue by silica gel chromatography eluting with 0-100%
dichloromethane in THF . Evaporate the product containing fractions with ethyl
acetate/heptane to give the title compound as a fine beige powder (106 g, 70%, 95%
purity). ES/MS: m/z 332.0 [M+H], [ ]D
20 = +42.11 ° (C= 0.532, chloroform).
Preparation 29
5-(1 -1,2,4-Triazol-1-yl)pyrazine-2-carboxylic acid
Stir a mixture of methyl 5-chloropyrazine-2-carboxylate (124 g, 718.55 mmol),
lH-l,2,4-triazole (198.5 g, 2874.2 mmol) and potassium carbonate (297.92 g, 2155.6
mmol) m N,N-dimethylformamide ( 1 L) at 100 °C for 15 hours. Cool to ambient
temperature and pour into water (2 L). Adjust the pH of the solution to 2-3 using
concentrated aqueous hydrochloric acid (about 500 mL) and stir for 30 minutes. Collect
the resulting solid by filtration and wash with water. Add water (500 mL) and ethanol
(500 mL), heat to 50-60 °C for 4 hours, and cool to ambient temperature. Collect the
solids by filtration and dry under vacuum at 40 °C to give the title compound as a white
solid. ES/MS: m/z 190.0 (.VI - ).
Preparation 30
N-[3-[(4aS,5S,7aS)-2-Benzamido-5-(l,l-difluo
d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-( 1,2,4-triazol- 1~yl)pyrazme~2-earboxamide
Scheme 3, step A : Add together N-[(4as,5s,7as)-7a-(5-amino-2-fluoro-phenyl)-5-
(l,l-difluoroethyi)~4,4a,5,7-tetrahydrofiiro[3,4~d][l ,3]thiazin-2-yl]benzamide (0.139 g,
0.32 mmol), 5-(lH-l,2,4-triazol-l-yl)pyrazrne-2-carboxylic acid (0.0852 g, 0.45 mmol),
and HOAt (0.0575 g, 0.41 mmol) in dichloromethane (4 ml): dimethylformamide ( 1 mL).
Add N,N-diisopropylethylamine (0.1 1 mL, 0.63 mmol) to the solution followed by EDCI
(0.079 g, 0.4 mmol) in one portion. Stir the reaction mixture at ambient temperature for
8 hours. Dilute the solution with ethyl acetate, wash with water and brine, and separate
the phases. Extract with ethyl acetate. Combine the organic extract and dry over
magnesium sulfate. Filter the solution and concentrate under reduced pressure to give a
residue. Purify the residue by silica gel chromatography, eluting with ethyl acetate:
dichloromethane (0-30% gradient), to give the title compound (0. 140 g, 59%). ES/MS
m z 609 (M+H).
Preparation 30a
N-[3-[(4aS,5S,7aS)-2-Benzamido-5-[cye!opropyK
te xahydro i [ ,4-d] [ ,3]thiazin-7a-y¾
2-carboxamide
Scheme 3, step A: Add together N-[(4aS,5S,7aS)-7a-(5-amino-2-fluoro-phenyl)-
5-[cyclopropyl(difluoro)methyl]-4,4a,5,74e1rahydrofuro[3,4-d][L3]thiazm-2-
yljbenzamide (21 mg, 0.045 mmol), 5-(lH-l,2,4-triazol-l-yl)pyrazine-2-carboxylic acid
(10 mg, 0.054 mmol), and HOBT (10 mg, 0.059 mmol) in dichloromethane (2.5 ml):
dimethylformamide (0.5 mL). Add N,N-diisopropylethylarnine (0.016 mL, 0.091 mmol)
to the solution followed by EDC1 ( 11 mg, 0.059 mmol) in one portion. Stir the reaction
mixture at ambient temperature for 18 hours. Dilute the solution with ethyl acetate,
water, and 1N NaOH (0.5 mL) and extract with ethyl acetate (3*). Combine the organic
extracts and dry over sodium sulfate. Filter the solution and concentrate under reduced
pressure to give a residue. Purify the residue by silica gel chromatography, eluting with
ethyl acetate: dichloromethane (0-100% gradient), to give the title compound (20 mg,
69%). ES/MS m z 635 (M+H).
Example 1
N-[3-[(4aS,5S,7aS)-2-Amino-5-iU-dM^
d] [1,3] thiazin-7a-yl]-4-fluoro-phenyl]-5-( 2,4-tri azol-1-yl)pyrazine-2-carboxamide
Scheme 3, step B: Heat a mixture of N-[3-[(4aS,5S,7aS)-2-benzamido-5-(l,ldifSuoroethyl)-
4,4a,5,7-tetrahydrofuro[3,4-d][l ,3]thiazm-7a-yl]-4-fluoro-phenyl]-5-
(l,2,4-triazol-l-yl)pyrazme-2-carboxamide (0.1148 g, 0.189 mmol), Omethylhydroxyiamine
hydrochloride (0.1575 g, 1.886 mmol), and pyridine (0.15 mi,
1.886 mmol) in THF (2 mL) and ethanoi (2 mL) at 45 °C for 5 hours. Cool the reaction
mixture to ambient temperature and stir for 2 days. Concentrate the solution under
reduced pressure to give a residue. Purify the residue by silica gel chromatography,
eluting with 7 N NH3 in methanol: diehloromethane (0-3% gradient), to give the title
compound (0.086 g, 90%). ES/MS m 505 (M+H).
Alternative Preparation Example 1
Scheme 4 Step D: Stir (4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(l,ldifluoroethyl)-
4a,5,7,7a-te1xahydro-4H-fuiO[3,4-d][l,3]thiazin-2~amine (96.5 g, 291
mmol) in ethyl acetate ( 1 L) under a nitrogen atmosphere at 50 °C and add 5-(lH-l,2,4-
triazol-l-yl)pyrazine -2-carboxylic acid (84 g, 439.45 mmol) slowly to the wann solution.
Stir for 10 minutes and add T3P® (1.67 M in ethyl acetate, 350 mL, 585 mmol) and stir at
50 °C for 17 hours. Cool to ambient temperature, dilute with diehloromethane ( 1 L) and
stir while quenching with a solution of sodium carbonate in water ( 8 g, 1.21 mol in 1
L). Dilute with diehloromethane ( 1 L) and water (2 L) and stir vigorously for 1 hour.
Filter through diatomaceous earth and wash with diehloromethane (3 500 mL),
methanol (500 mL), water (500 mL), aqueous saturated sodium bicarbonate (500 mL),
and : methanol:dichloromethane (6 500 mL). Separate the layers and extract the
aqueous with dichloromethane (3 1 L). Combine all organic phases and evaporate to
give a residue. Sonicate the residue in dichloromethane ( 1 L) for 15 minutes and collect
the solids by filtration washing with dichloromethane (5 200 niL). Add saturated
aqueous sodium hydrogen carbonate until pH 8 is obtained and stir vigorously with
dichloromethane ( 1 L) and methanol (500 m ). Remove the solids by filtration and
extract the filtrate with dichloromethane (2 500 mL). Dissolve the solids with
dichloromethane:methanol (1:1, 500 mL) and combine this solution with the other
organic phases. Remove the solvent under reduced pressure adding dichloromethane to
maintain a solution and then once a final volume of about 300 m is obtained, purify the
solution by silica gel chromatography, eluting with 5% of 0 3 M ammonia/methanol in
dichloromethane to give a light brown solid. Dissolve the solid in hot ethanol (2.5 L),
filter while hot, and cool to ambient temperature over 1 hour. Collect the solids by
filtration and wash with ethanol (2 x 250 mL) and dry under vacuum. Evaporate the
filtrate to dryness and further purify by silica gel chromatography eluting first with 65%
ethyl acetate in 50: 1 iso-hexane /7 N ammonia in methanol then 50:1 ethyl acetate/7 N
ammonia in methanol. If required, further purification can be completed by SFC,
column: Chiralpak AD-H (5 ), 50 250 mm; eluent: 35% isopropanol (0.2%
diethylmethylamine) in CO?; flow rate: 300 g/minute at IJV 220 nm. After evaporation
and vacuum drying, slurry the material in ethanol (1.5 L) and stir with gentle wanning
between (36 and 45 °C) for 20 minutes. Collect the solid by filtration washing with
ethanol (100 mL). Further material can be recovered from the filtrate; evaporate to
dryness, reflux in ethanol, remove the solids by hot filtration and then cool the filtrate to
ambient temperature. Collect solids by filtration, washing with ethanol and combine with
the material obtained from the above filtration. Dry the combined solids under vacuum at
40 °C to give the title compound as a white solid ( 103.3 g, 68%, containing 2.5 wt%
ethanol). ES/MS m/z 505.0 (M+H), [a]D
20 +149.4 ° (C= 1, chloroform).
Example 1A
-[3-[(4aS,5S,7aS)-2-amino-5-(l ,l -di uoroethy l)-4,4a,5,7-tetrahydrofuro[3 4-
d][l ,3]thiazin-7a-yl]-4-fl uoro-phe yl] 4 -
methylbenzenesul fonate
Dissolve N-[3-[(4aS,5S,7aS)-2-amino-5-(l,l-difluoroethyl)-4,4a,5,7-
tetrahydrofuro[3,4-d] [1,3]thiazin-7a-yl] -4-fiuoro-pheiiyl]-5-( 1,2,4-triazoi- 1-yl)pyrazine-
2-carboxamide (600 mg, 1. 89 mmol) in acetone (9 mL)and water ( 1 niL). Heat the
resulting suspension to 60 °C. Add /?-toluenesulfonic acid monohydrate (420 mg, 2.208
mmol) dissolved in acetone ( 1 niL). Stir the mixture overnight at 60 °C. Cool the
mixture to room temperature, filter the solids by vacuum and wash with acetone ( 1 mL)
and air dry overnight to give the title compound (743 mg, 73%).
X-Ray Powder Diffraction (XRD)
The XRD patterns of crystalline solids are obtained on a Bruker D4 Endeavor Xray
powder diffractometer, equipped with a CuKa source = 1.54060 A and a Vantec
detector, operating at 35 kV and 50 mA. The sample is scanned between 4 and 40° in 2,
with a step size of 0.009° in 2, a scan rate of 0.5 seconds/step, with 0.6 mm divergence,
5.28 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. The crystal form
diffraction patterns are collected at ambient temperature and relative humidity it is well
known in the crystallography art that, for any given crystal forrn, 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 United States Pharmacopeia #23, National Formulary # 8,
pages 1843-1844, 1995. 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 2will take into
account these potential variations without hindering the unequivocal identification of the
indicated eiystal form. Confirmation of a crystal form may be made based on any unique
combination of distinguishing peaks (in units of ° 2), typically the more prominent
peaks. The crystal form diffraction patterns, collected at ambient temperature and relative
humidity, were adjusted based on NIST 675 standard peaks at 8.853 and 26.774 degrees
2-theta.
A prepared sample of crystalline N-[3-[(4aS,5S,7aS)-2-amino-5-(l,ldifluoroe1hyl)-
4,4a,5,7-telTahydrofuro[3,4-d][l,3]thiazm-7a-yl]-4-f]uoro-ph
(l,2,4-triazol-l-yl)pyrazme-2-carboxamide 4-methylbenzenesulfonate is characterized by
an XRD pattern using Cu a radiation as having diffraction peaks (2-theta values) as
described in the Table below. Specifically, the pattern contains a peak at 17.3° in
combination with one or more of the peaks selected from the group consisting of 14.8,
12.7, and 4.9; with a tolerance for the diffraction angles of 0.2 degrees.
Table 1 : X-ray powder diffraction peaks of Example 1A.
Example B
N-[3-[(4aS,5S,7aS)~2~armno-5-(i,!-difiuoroe%
d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(l ,2,4-triazol-l -yl)pyrazine-2-carboxamide
malonate
Add together N-[3-[(4aS,5S,7aS)-2-amino-5-(l, l-difluoroethyl)-4,4a,5,7-
tetrahydrofuro[3,4-d] [1,3]thiazin-7a-yl] -4-fluoro-phenyl] -5-( 1,2,4-triazoi- 1-yl)pyrazine-
2-carboxamide (201 mg, 0.398 mmol) and malonic acid (104 mg, 0.999 mmol)in 95%
ethanol-water ( 5 mL). Stir the mixture 65 °C until a solution a clear solution is obtained.
A thick white solid precipitates after a few minutes. Stir the suspension for 1 hour at 55
° and then cool to room temperature with stirring. Filter the solids under vacuum and
air dry for 2 days to give the title compound (477 mg, 80%).
A prepared sample of crystalline N-[3-[(4aS,5S,7aS)-2-amino-5-(l,ldifluoiOethy2)-
4,4a,5,7-tetrahydromro[3,4-d][l 3]thiazm-7a-yl]-4-fluoiO-phenyi]-5-
(l,2,4-triazol-l-y])pyrazme-2-carboxamide malonate is characterized by an XRD pattern
using CuKa radiation as having diffraction peaks (2-theta values) as described in Table 2
below. Specifically, the pattern contains a peak at 22.7 in combination with one or more
of the peaks selected from the group consisting of 16.8, 17.2, and 24.0; with a tolerance
for the diffraction angles of 0.2 degrees.
Table 2 : X-ray powder diffraction peaks of Example IB.
Angle Relative Intensity
Peak
(°2-Theta +/- 0.2°) (% of most intense peak)
1 5.5 39
10.3 44
3 11.8 55
4 15.3 3
5 16.8 62
6 17.2 57
7 18.3 4 1
8 22.4 60
9 22.7 100
10 24.0 53
Example 1C
N-[3-[(4aS,5S,7aS)-2-arrdno-5-(l,l-difluoroe1hyl)-4,4a,5,7-tetrahy(kofuro[3,4-
d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-(l ,2,4-triazol-l -yl)pyrazme-2-carboxamide
h drate
Suspend N-[3~[(4aS,5 S,7aS)-2-amino-5 -(1,1-difluoroethyl)~4,4a,5 ,7-
tetrahydrofuro[3,4-d] [ ,3]thiazin-7a~yl] -4-f! uoro-phenyl] -5-( ,2,4-triazol-l -yl)pyrazine-
2-carboxamide (116 mg, 0.23mmol ) in 1:1 THF:water (2 mL) at 70 °C. Stir the solution
for at least 2 days, filter the solid, and dry under a nitrogen stream to give the title
compound
A prepared sample of N-[3-[(4aS,5S,7aS)-2-amino-5-(l,l~difiuoroethyl)-4,4a,5,7-
tetrahydrofmO[3,4-d] [ ,3] hiazm
2-carboxamide hydrateis characterized by an XRD pattern using CuKa radiation as
having diffraction peaks (2-theta values) as described in Table 3 below. Specifically, the
pattern contains a peak at 13.0 in combination with one or more of the peaks selected
from the group consisting of 7.8, 10.5, 11.0, 14.9, 19.7, 21.3, and 26.9 with a tolerance
for the diffraction angles of 0.2 degrees.
Table 3. X-ray powder diffraction peaks of Example 1C.
Example 2
N-[3-[(4aS,5S,7aS)~2-Amino~5~(l,l-difluoropropyl)-4,4a,5,7-tetrahydrofiiro[3,4-
d][l,3]tMazin-7a-y2]-4-fluoro-pheny2]-5-(l,2,4-triazol-l-yl)pyrazine-2-carboxaniide
Scheme 3, steps A and B: Add together 5-(lH-l,2,4-triazol-l-yl)pyrazine-2-
carboxylic acid (0.1 6 g, 0.608 mmol), acetonitrile (4.05 mL), dimethyl formamide
(0.00314 mL), and then oxalyl chloride (0.154 g, 0.105 mL, 1.22 mmol) dropwtse under
nitrogen. Stir the reaction for 30 minutes and then concentrate in vacuo. Dissolve the
residue in acetonitrile (4.05 mL,) and add dropwise to a mixture of (4aS,5S,7aS)-7a-(5-
amino-2-fluorophenyl)-5-(l ,1-difluoropropyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-
d][L3]thiazin-2 -amine (0.140 g, 0.405 mmol) in ethanol (4.05 mL) and water (1.35 mL)
with stirring. Upon complete addition, dilute the reaction with chloroform and wash with
saturated sodium bicarbonate solution. Dilute the organics with methanol and add to an
SCX-2 cartridge. Flush the SCX-2 cartridge with one column volume of methanol and
discard, and then flush the SCX-2 cartridge with one column volume of 7 M methanolic
ammonia. Concentrate the methanolic ammonia flush in vacuo and purify by silica gel
chromatography, eluting with dichioromethane/methanol (100-85% gradient) to give a
residue. Further purify by achiral SFC (Supercritical Fluid Chromatography) (Column:
benzenesulfonamide (BzS) (5 ), Princeton Chromatography, 21.2 x 250 mm; eluent:
22% methanol (1% 2 M ammonia in methanol) i CC¾ flow rate; 70 mL/rninute at UV
250 nm; back pressure; 100 bar; temperature: 40 °C). Solubilize the residue in
chloroform and wash with brine. Pass the organics through a hydrophobic frit and
concentrate in vacuo to give the title compound (0.0658 g, 0.127 mmol, 31%). ES/MS
m z 519 (M+H).
Example 3
N-[3-[(4aS,5 S,7aS)-2-Amino-5 -[eyclopropyl(difluoro)methyl] -4,4a,5 ,7-
te1xahydro&ro[3,4-d][l,3]thiazin-7a-yl]-4-fluoro-pheny2]-5-(l,2,4-triazol-l-yl)p^^
2-carboxamide
Scheme 3, step B: Heat a mixture of N-[3-[(4aS,5S,7aS)-2~benzamido-5-
[cyclopropyl(difluoro)methyl]-4,4a,5,7-te1xahydrofuro[3,4-d][l,3]thiazin-7a-yl]-4-fluorophenyi]-
5-(l,2,4-triazo2-l-yi)pyrazine-2-carboxamide (20 mg, 0.0315 mmol), Omethyihydroxylamme
hydrochloride (26 mg, 0.3 5 mmol), and pyridine (0.026 ml, 0.315
mmol) in ethanol (3 m ) at 55 °C for 8 hours. Cool the reaction mixture to ambient
temperature and concentrate the solution under reduced pressure to give a residue. Purify
the residue by silica gel chromatography, eluting with 7N N¾ in methanol:
dichloromethane (0.5-10% gradient), to give the title compound (15 g, 90%). ES/MS
m/ 531 (M+H).
In vitro Assay Procedures:
To assess selectivity of BACE1 over BACE2, the test compound is evaluated in
FRET-based enzymatic assays using specific substrates for BACE1 and BACE2 as
described below. For in vitro enzymatic and cellular assays, the test compound is
prepared in DMSO to make up a 10 mM stock solution. The stock solution is serially
diluted in DMSO to obtain a ten-point dilution eun'e with final compound concentrations
ranging from 10 to 0.05 nM in a 96-well round-bottom plate before conducting the in
vitro enzymatic and whole cell assays.
In vitro protease inhibition assays:
Expression of AwBACEl:Fc and AwBACE2:Fc.
Human BACE1 (accession number: AF190725) and human BACE2 (accession
number: AF 204944) are cloned from total brain cDNA by RT-PCR. The nucleotide
sequences corresponding to amino acid sequences # 1 to 460 are inserted into the cDNA
encoding human TgG (Fc) polypeptide (Vassar et al, Science, 286, 735-742 (1999)).
This fusion protein of BACE1(1 -460) or BACE2( 1-460) and human Fc, named
AwBACEl:FC and AwBACE2:Fcrespectively, are constructed into the pJB02 vector.
Human BACEl(l-460):Fc (AwBACEl:Fc) and human BACE2(l-460):Fc (AwBACE2:Fc)
are transiently expressed in HEK293 cells. 250 , g cDNA of each construct are mixed
with Fugene 6 and added to 1 liter HEK293 cells. Four days after the transfection,
conditioned media are harvested for purification. AwBACEl:Fc and AwBACE2:Fcare
purified by Protein A chromatography as described below. The enzymes are stored at -
80 °C in small aiiquots. (See Yang, et. al, J. Neurochemistry, 91(6) 1249-59 (2004),
Purification of AwBACEl:Fc and AwBACE2:Fc.
Conditioned media of HEK293 cell transiently transfected wit AwBACEl:Fc or
AwBACE2:FccDNA are collected. Cell debris is removed by filtering the conditioned
media through 0.22 sterile filter. 5 ml Protein A-agarose (bed volume) is added to 4
liter conditioned media. This mixture is gently stirred overnight at 4 °C. The Protein Aagarose
res n is collected and packed into a low-pressure chromatography column. The
column is washed with 20x bed volumes of PBS at a flow rate 20 ml per hour. Bound
BACEl :Fc or A«BACE2:Fc protein is eluted with 50 M acetic acid, p 3.6, at flow
rate 20 ml per hour. 1ml fractions of eluent are neutralized immediately with 0.5 ml 200
mM ammonium acetate, pH 6.5. The purity of final product is assessed by
electrophoresis in 4-20% Tris-Glycine SDS-PAGE. The enzyme is stored at -80 °C in
small aiiquots.
BACEl FRET Assay
Serial dilutions of the test compound are prepared as described above. The
compound is further diluted 20 in KH2PO4 buffer. Ten uL of each dilution is added to
each well on row A to H of a corresponding low protein binding black plate containing
the reaction mixture (25 uL of 50 mM 2PG4, pH 4.6, 1mM TRITON® X-100, 1
mg mL BSA, and 15 of FRET substrate based upon the sequence of APP) (See Yang,
et. ah, J. Neurochemistry, 91(6) 1249-59 (2004)). The content is mixed well on a plate
shaker for 10 minutes. Fifteen uL of two hundred pM human BACEl (l-460):Fc (See
Vasser, et a , Science, 28 , 735-741 (1999)) in the KH2PO4 buffer is added to the plate
containing substrate and the test compound to initiate the reaction. The RFU of the
mixture at time 0 is recorded at excitation wavelength 355 nm and emission wavelength
460 nm, after brief mixing on a plate shaker. The reaction plate is covered with
aluminum foil and kept in a dark humidified oven at room temperatui e for 16 to 24 hours.
The RFU at the end of incubation is recorded with the same excitation and emission
settings used at time 0. The difference of the RFU at time 0 and the end of incubation is
representative of the activity of BACEl under the compound treatment. RFU differences
are plotted versus inhibitor concentration and a curve is fitted with a four-parameter
logistic equation to obtain the IC50 value. (May, et ah, Journal ofNeuroscience, 3 1.
16507-16516 (2011)).
The compound of Example 1 herein is tested essentially as described above and
exhibits an C50 for BACEl of 1.19 nM ± 0.48, n= (Mean ± SEM; SEM = standard error
of the mean). This data demonstrates that the compound of Example 1 inhibits purified
recombinant BACEl enzyme activity in vitro.
BACE2 TMEM27 FRET Assay
Transmembrane protein 27 (TMEM27) (Accession Number NM_020665), also
known as Collectrin) is a recently described substrate for BACE2, but not BACE1
(Esterhazy, etal, Cell Metabolism, 14, 365-377 (201 )). To evaluate the test compound
for inhibition of BACE2 enzymatic activity, a FRET peptide (dabcyl-QTLEFLKIPSLucY)
based upon the amino acid sequence of human TMEM27 is used as a substrate
(Esterhazy, et al, Cell Metabolism, 14, 365-377 (201 1)). Serial dilutions of the test
compound are prepared as described above. The compound is further diluted 20x in
24 buffer. Ten L of each dilution is added to each well on row A to of a
corresponding low protein binding black plate containing the reaction mixture (25 of
50 mM KH2PO4, pH 4.6, 1 M TRITON® X-100, 1mg mL BSA, and 5 of TMEM
FRET substrate). Fifteen uL of twenty human BACE2 (l-460);Fe (See Vasser, et ah,
Science, 286, 735-741 (1999)) in KH2PO4 buffer is then added to the plate containing
substrate and the test compound to initiate the reaction. The conten t is mixed well on a
plate shaker for 10 minutes. The RFU of the mixture at time 0 is recorded at excitation
wavelength 430 ran and emission wavelength 535 rail The reaction plate is covered with
aluminum foil and kept in a dark humidified oven at room temperature for 16 to 24 hours.
The RFU at the end of incubation is recorded with the same excitation and emission
settings used at time 0. The difference of the RFU at time 0 and the end of incubation is
representative of the activity of BACE2 under the compound treatment. RFU differences
are plotted versus inhibitor concentration and a curve is fitted with a four-parameter
logistic equation to obtain the IC50 value. (May, et ah, Journal ofNeuroscience, 3 1,
1650746516 (2011)).
The compound of Example 1 herein is tested essentially as described above and
exhibits a BACE2 IC 0 of 479 nM ± 202, n=4(Mean ± SEM; SEM === standard error of the
mean). The ratio of BACE1 (FRET IC50 enzyme assay) to BACE2 (TMEM27 FRET IC50
assay) is about 400-fold, indicating functional selectivity for inhibiting the BACE1
enzyme. The data set forth above demonstrates that the compound of Example 1 is
selective for BACE1 over BACE2.
SH-SY5YAPP695Wt Whole Cell Assay
The routine whole cell assay for the measurement of inhibition of BACE1 activity
utilizes the human neuroblastoma cell line SH-SY5Y (ATCC Accession No. CRL2266)
stably expressing a human APP695Wt cDNA. Cells are routinely used up to passage
number 6 and then discarded.
SH-SY5YAPP695Wt cells are plated in 96 well tissue culture plates at 5.0* 104
cells/well in 200 \i culture media (50% MEM/EBSS and Ham's F12, 1 x each sodium
pyruvate, non-essential amino acids and Na bicarbonate containing 10% FBS). The
following day, media is removed from the cells, fresh media added then incubated at 37
°C for 24 hours in the presence/absence of test compound at the desired concentration
range.
At the end of the incubation, conditioned media are analyzed for evidence of betasecretase
activity by analysis of Abeta peptides 1-40 and 1-42 by specific sandwich
ELISAs. To measure these specific isoforms of Abeta, monoclonal 2G3 is used as a
capture antibody for Abeta 1-40 and monoclonal 2 1F12 as a capture antibody for Abeta
1-42. Both Abeta 1-40 and Abeta 1-42 ELISAs use biotinylated 3D6 as the reporting
antibody (for description of antibodies, see Johnson-Wood, et ah, Proc. Natl. Acad. Sci.
USA 94, 1550-1 555 ( 1997)). The concentration of Abeta released in the conditioned
media following the compound treatment corresponds to the activity of BACE1 under
such conditions. The 10-point inhibition curve is plotted and fitted with the fourparameter
logistic equation to obtain the IC50 values for the Abeta-lowering effect. The
compound of Example 1 is tested essentially as described above and exhibits the
following activity for Abeta-lowering as shown in table 4
Table 4
(Mean ± SEM; SEM = standard error of the mean)
In vivo Inhibition of Beta-Secretase
Several animal models, including mouse, guinea pig, dog, and monkey, may be
used to screen for inhibition of beta-secretase activity in vivo following compound
treatment. Animals used in this invention can be wild type, transgenic, or gene Imockout
animals. For example, the PDAPP mouse model, prepared as described in Games et a ,
Nature 373, 523-527 (1995), and other non-transgenic or gene knockout animals are
useful to analyze in vivo inhibition of Abeta and sAPPbeta production in the presence of
inhibitory compounds. Generally, 2 month old PDAPP mice, gene knockout mice or nontransgenic
animals are administered compound formulated in vehicles, such as com oil,
beta-cyclodextran, phosphate buffers, PHARMASOLVE®, or other suitable vehicles via
oral, subcutaneous, intra-venous, feeding, or other route of administration. One to
twenty-four hours following the administration of compound, animals are sacrificed, and
brains are removed for analysis of Abeta 1-x. "Abeta 1-x" as used herein refers to the
sum of Abeta species that begin with residue 1 and end with a C-terrninus greater than
residue 28. This detects the majority of Abeta species and is often called "total Abeta".
Total Abeta peptides (Abeta 1-x) levels are measured by a sandwich ELISA, using
monoclonal 266 as a capture antibody and biotinylated 3D6 as reporting antibody. (See
May, et al., Journal ofNeuroscience, 31, 16507-16516 (20 1)).
For acute studies, compound or appropriate vehicle is administered and animals
are sacrificed at about 3 hours after dosing. Brain tissue, is obtained from selected
animals and analyzed for the presence of Abeta 1-x. After chronic dosing brain tissues of
older APP transgenic animals may also be analyzed for the amount of beta-amyloid
plaques following compound treatment.
Animals (PDAPP or other APP transgenic or non-transgenic mice) administered
an inhibitor}' compound may demonstrate the reduction of Abeta in brain tissues, as
compared with vehicle-treated controls or time zero controls. For example, a 3, 10, and
30 mg/kg oral dose of Example 1, to young female PDAPP mice reduced Abeta 1-x
peptide levels in brain hippocampus by 23% (non-significant), 43% (p<0.05), and 58%
(p<0.01), respectively. In brain cortical tissue, doses of 3, 10, and 30 mg/kg of Example
1 reduced Abeta 1-x levels by 43%, 59%, and 73% (all values p<0.01) compared to
vehicle-treated mice three hours after dosing.
Given the activity of the Example 1, against the BACEl enzyme in vitro, these
Abeta- lowering effects are consistent with BACE inhibition in vivo, and further
demonstrate CNS penetration of Example 1.
These studies show that compound of the present invention inhibits BACEl and
is, therefore, useful in reducing Abeta levels.
WE CLAIM:
1. A compound of the formula:
wherein R is methyl, ethyl, or cyclopropyl;
or a pharmaceutically acceptable salt thereof.
2. The compound or salt thereof according to claim 1 wherein R is methyl.
3. The compound or salt thereof according to either claim 1 or claim 2 wherein
the compound is:
The compound or salt thereof according to any one of claims 1-3 wherein
compound is:
5. The compound accordin to any one of claims 1-4 which is:
6. The salt acc rding to any one of claims 1-4 which is:
7. The salt accordin to any one of claims 1-4 which is:
8. The compound accordin to any one of claims 1-4 which is:
9. The compound or salt thereof according to any one of claims 1-4 wherein the
compound is N-[3-[(4aS,5S,7aS)-2-amino-5-(Ll-difluoroethyl)-4,4a,5,7-
tetrahydrofuro [3,4-d] [ ,3 ]thiazin-7a-yl]-4-fluoro-phenyl]-5 -( ,2,4-triazol -1-
yl)pyrazine-2-carboxamide.
10. The compound according to claim 5 which is N-[3-[(4aS,5S,7aS)-2-amino-5-
( 1,1-difluoroethy3)-4,4a,5,7-tetrahydrofuro[3,4-d] [1,3]thiazin-7a-yl]-4-fluorophenyl]-
5-(l,2,4-triazol-l-yl)pyrazine-2-carboxamide.
11. The compound according to any one of claims 1-4 which is N-[3-
[(4aS,5 S,7aS)-2-amino-5 -(1,1-difluoroethyl)-4,4a,5 ,7-tetrahydrofuro [3Ad]
[1,3]thiazin-7a-yl]-4-fluoro-phenyl] -5-(1,2,4-triazol- 1-yl)pyrazine-2-
carboxamide hydrate.
12. The salt according to any one of claims 1-4 which is N-[3-[(4aS,5S,7aS)-2-
amino-5-( 1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d] [1,3]thiazin-7a-yl]-
4-fluoro-phenyl]-5-(l,2,4-triazo]-l-yl)pyrazme-2-carboxamide 4-
methylbenzenesulfonate.
13. The salt according to any one of claims 1-4 which is N-[3-[(4aS,5S,7aS)-2-
amino-5-( 1, -difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d] [1,3]thiazin-7a-yl]-
4-fluoro-phenyl]-5-(l,2,4-triazol-l-y])pyrazme-2-carboxamide malonate.
14. A method of treating Alzheimer's disease in a patient, comprising
administering to a patient in need of such treatment an effective amount of a
compound of any one of claims 1-13, or a pharmaceutically acceptable salt
thereof.
15. A method of treating the progression of mild cognitive impairment to
Alzheimer's disease in a patient, comprising administering to a patient in need
of such treatment an effective amount of a compound of any one of claims 1-
13, or a pharmaceutically acceptable salt thereof.
16. A compound or pharmaceuticaliy acceptable salt thereof according to any one
of claims 1-13 for use in therapy.
1 . A compound or pharmaceutically acceptable salt thereof according to any one
of claims 1-13 for use in the treatment of Alzheimer's disease.
18. A compound or pharmaceutically acceptable salt thereof according to any one
of claims 1-13 for use in treating the progression of mild cognitive impairment
to Alzheimer's disease.
19. A pharmaceutical composition, comprising a compound or a pharmaceutically
acceptable salt thereof according to any one of claims 1-13 with one or more
pharmaceutically acceptable carriers, diluents, or excipients.
20. A process for preparing a pharmaceutical composition, comprising admixing a
compound or a pharmaceutically acceptable salt thereof according to any one
of claims 1-13 with one or more pharmaceutically acceptable carriers,
diluents, or excipients.