The present invention relates to novel crystalline BACE inhibitors, to
pharmaceutical compositions comprising the crystalline BACE inhibitors, to methods of
using the crystalline BACE inhibitors to treat physiological disorders, and to
intermediates and processes useful in the synthesis thereof.
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, 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.
United States Patent No. 8,158,620 discloses fused aminodihydrothiazine
derivatives which possess BACE 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, J. Neuroscience, 31(46), pages 16507-16516
(201 1) discloses (S)-4-(2,4-difluoro-5-pyrimidin-5-yl-phenyl)-4-methyl-5,6-dihydro-4 H-
[l,3]thiazin-2-ylamine, an orally administered CNS-active BACE inhibitor.
Crystalline forms of BACE inhibitors are desired to provide for ease of
preparation of pharmaceutical formulations and for pharmaceutical compositions with
improved stability.
Accordingly, the resent invention provides a compound of Formula I :
Formula I
which is crystalline.
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 the compound of Formula I which is crystalline. The present invention further
provides a method of preventing 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 the compound of Formula I which is crystalline. 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 the compound
of Formula I which is crystalline. The present invention also provides a method for
inhibiting BACE-mediated cleavage of amyloid precursor protein, comprising
administering to a patient in need of such treatment an effective amount of the compound
of Formula I which is crystalline. 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 the compound of Formula I which is crystalline.
Furthermore, this invention provides a compound of Formula I which is
crystalline for use in therapy, in particular for the treatment of Alzheimer's disease or for
the prevention of the progression of mild cognitive impairment to Alzheimer's disease.
Even furthermore, this invention provides the use of the compound of Formula I which is
crystalline for the manufacture of a medicament for the treatment of Alzheimer's disease
or for the prevention of the progression of mild cognitive impairment to Alzheimer's
disease.
The invention further provides a pharmaceutical composition, comprising the
compound of Formula I which is crystalline with one or more pharmaceutically
acceptable carriers, diluents, or excipients. This invention also encompasses novel
intermediates and processes for the synthesis of the compound of Formula I which is
crystalline.
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 a , Arch. Neurol, 58, 397-405 (2001); Petersen, et a , Arch.
Neurol, 56, 303-308 (1999)). The term "prevention of the progression of mild cognitive
impairment to Alzheimer's disease" includes slowing, arresting, 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. In 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, transdermal, and parenteral routes. Most preferably, such
compositions are for oral administration or transdermal, with oral administration be
especially preferred. 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 crystalline compounds of Formula I are particularly useful in the treatment
methods of the invention, but certain forms are preferred. The following paragraphs
describe such forms. It will be understood that these preferences are applicable both to
the treatment methods and to the new compounds of the invention.
Cyrstalline Form lof N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-
4,4a,5 ,7-tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl] -5-methoxypyrazine-
2-carboxamide;
Crystalline Form 2 of N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-
4,4a,5 ,7-tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl] -5-methoxypyrazine-
2-carboxamide;
Crystalline Form 2 of N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-
4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-methoxypyrazine-
2-carboxamide which is characterized by a substantial peak in the X-ray
diffraction spectrum, at diffraction angle 2-theta of 11.8°, in combination with one or
more of the peaks selected from the group consisting of 18.6°, 19.3°, and 26.7°; with a
tolerance for the diffraction angles of 0.2 degrees;
Crystalline Form 3 of N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-
4,4a,5 ,7-tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl] -5-methoxypyrazine-
2-carboxamide; and
Crystalline Form 3 of N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-
4,4a,5 ,7-tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl] -5-methoxypyrazine-
2-carboxamide which is characterized by a substantial peak in the X-ray
diffraction spectrum, at diffraction angle 2-theta of 15.7° in combination with one or
more of the peaks selected from the group consisting of 18.1°, 27.0°, and 19.7°; with a
tolerance for the diffraction angles of 0.2 degrees, are preferred.
Crystalline Form 2 of N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-
4,4a,5 ,7-tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl] -5-methoxypyrazine-
2-carboxamide, is particularly preferred.
Crystalline Form 2 of N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-
4,4a,5 ,7-tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl] -5-methoxypyrazine-
2-carboxamide, which is characterized by a substantial peak in the X-ray
diffraction spectrum, at diffraction angle 2-theta of 11.8°, in combination with one or
more of the peaks selected from the group consisting of 18.6°, 19.3°, and 26.7°; with a
tolerance for the diffraction angles of 0.2 degrees, is especially preferred.
One of ordinary skill in the art will appreciate that compounds of the invention
can exist in tautomeric forms, as depicted in Scheme A. 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 A
Additionally, certain intermediates described in the following schemes may
contain one or more nitrogen protecting groups. The variable protecting group may be
the same or different in each occurrence 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).
Certain stereochemical centers have been left unspecified and certain substituents
have been eliminated in the following schemes for the sake of clarity and are not intended
to limit the teaching of the schemes in any way. Furthermore, individual isomers,
enantiomers, and diastereomers may be separated or resolved by one of ordinary skill in
the art at any convenient point in the synthesis of compounds of the invention, by
methods such as selective crystallization techniques or chiral chromatography (See for
example, J. Jacques, et al., "Enantiomers, Racemates, and Resolutions", John Wiley and
Sons, Inc., 1981, and E.L. Eliel and S.H. Wilen," Stereochemistry of Organic
Compounds", Wiley-Interscience, 1994). The designations "isomer 1" and "isomer 2"
refer to the compounds that elute from chiral chromatography first and second,
respectively, and if chiral chromatography is initiated early in the synthesis, the same
designation is applied to subsequent intermediates and examples.
Certain abbreviations are defined as follows: "APP" refers to amyloid precursor
protein; "CSF" refers to cerebrospinal fluid; "DCM" refers to dichloromethane; "DIPEA"
refers to diisopropylethylamine or N-ethyl-N-isopropyl-propan-2-amine; "DMEM" refers
to Dulbecco's Modified Eagle's Medium; "DMF" refers to dimethylformamide; "DMSO"
refers to dimethyl sulfoxide; "EDO" refers to l-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride; "ee" refers to enantiomeric excess; "EtOAc" refers to
ethyl acetate; "Ex" refers to example; "F12" refers to Ham's F12 medium; "FBS" refers
to Fetal Bovine Serum; "FRET" refers to fluorescence resonance energy transfer; "HEK"
refers to human embryonic kidney; "HOAc" refers to acetic acid; "HOBt" refers to 1-
hydroxylbenzotriazole hydrate; "HPLC refers to high-performance liquid
chromatography; "IC 50" refers to the concentration of an agent that produces 50% of the
maximal inhibitory response possible for that agent; "min" refers to minute or minutes;
"MTBE" refers to methyl rt-butyl ether; "PDAPP" refers to platelet derived amyloid
precursor protein; "Prep" refers to preparation; "RFU" refers to relative fluorescence unit;
"SCX" refers to strong cation exchange; "Rt" refers to retention time; and "THF" refers
to tetrahydrofuran.
The compounds of the present invention may be prepared by a variety of
procedures known in the art, some of which are illustrated in the Preparations and
Examples below. The specific synthetic steps for each of the routes described may be
combined in different ways, or in conjunction with steps from different preparations to
prepare compounds of the invention. The products of each step can be recovered by
conventional methods well known in the art, including extraction, evaporation,
precipitation, chromatography, filtration, trituration, or crystallization. The reagents and
starting materials are readily available to one of ordinary skill in the art.
Preparation 1
2-Bromo- 1-(5 -bromo-2-fluorophenyl)ethan- 1-
N-bromosuccinimide (984 g, 5.53 mol) is added portion-wise to a solution of l-(5-
bromo-2-fluorophenyl)ethan-l-one (1000 g, 4.6 mol) and />-toluene sulfonic acid (1315 g,
7.64 mol) in DCM (7 L) at 35 °C. The mixture is stirred and heated to 40 °C. The
mixture is cooled to 24 °C, and 7% NaHC0 3 (5 L) is added. The layers are separated and
the organic layer is washed with 10% Na2S0 3 (5 L) and water (5 L). The organic layer is
concentrated to 2-3 volumes to give the title compound which is used without further
purification.
Preparation 2
5-Allyl-6a-(5-bromo-2-fluorophenyl)- 1-(4-methoxybenzyl)hexahydro
pyrrolo[3,4-c]isoxazole
To a solution of 2-bromo-l-(5-bromo-2-fluorophenyl)ethan-l-one (1363 g, 4.61
mol) in toluene (10 L) is added diallylamine (537 g, 5.53 mol) and dipea (2381 g, 18.42
mol). The mixture is stirred for 4 hours at 40 °C to give l-(5-bromo-2-fluorophenyl)-2-
(diallylamino)ethan-l-one, which is not isolated. N-(4-methoxybenzyl)hydroxylamine
(847 g, 5.53 mol) and Ti(OiPr)4 (1965 g, 6.91 mol) are added to the mixture containing
crude l-(5-bromo-2-fluorophenyl)-2-(diallylamino)ethan-l-one. The mixture is stirred at
90 °C for 2 hours. The mixture is cooled to 20 °C, and 50% citric acid monohydrate (4 L)
and saturated Na2C0 3 (4 L) are added. The layers are separated and the aqueous is
extracted with MTBE (5 L). The organic extract is washed with water (5 L), and filtered
through diatomaceous earth and concentrated to dryness. EtOAc (10 L) and Oxalic acid
(580 g) are added to the residue and a solid is filtered and added to 1N NaOH (13 L).
MTBE (5 L) is added and the mixture is filtered through diatomaceous earth. The layers
are separated and the organic layer is concentrated to 2 volumes. Heptane (3 L) is added
and the solution is cooled to 10 °C. The resulting solid is filtered to give the title
compound (1330 g, 64%). l NMR(400 MHz, CDC13) 2.51-2.49(m, 3H), 3.09-3.04(m,
3H), 3.78-3.41(m, 6H), 4.01(m, 1H), 5.24-5.01(m, 2H), 5.89-5.85(m, 1H), 6.82-6.80(m,
2H), 7.51-7.13(m, 3H), 7.63-7.62(m, 1H), 7.65-7.64(m, 1H).
Preparation 3
5-Allyl-6a-(5 -bromo-2-fluorophenyl)hexahydro- 1H-pyrrolo [3,4-c]isoxazole
hydrochloride
Trifluoroacetic acid (4 L, 52.9 mol) is added drop wise to a solution of 5-allyl-6a-(5-
bromo-2-fluorophenyl)- 1-(4-methoxybenzyl)hexahydro- 1H-pyrrolo [3,4-c]isoxazole
(1990 g, 4.45 mol) in DCM (12 L) at a rate to maintain the temperature below 35 °C.
After the addition is complete, the mixture is warmed to 33-43 °C and stirred for 6 hours.
NaOH (20%, 10 L) is added at a rate to maintain the temperature below 35 °C. The
layers are separated and the organic layer is washed with water (6 L). The solution is
concentrated, ethanol (16 L) is added, and the mixture is filtered through diatomaceous
earth. The filtrate is concentrated and EtOAc (10 L) is added. 4 M HCl in EtOAc (8 L) is
added and the resulting solid is filtered and dried to give the title compound (1385 g,
85.6%). ES m/z 327.1 (M+l)
Preparation 4
(l-Allyl-4-arnino-4-(5-bromo-2-fluorophenyl)pyrrolidin-3-yl)metlianol
A saturated aqueous solution of sodium carbonate is added to a solution of 5-allyl-
6a-(5-bromo-2-fluorophenyl)hexaliydro-lH-pyrrolo[3,4-c]isoxazole hydrochloride (1400
g, 3.85 mol) in DCM (7 L) to reach a pH>9. The layers are separated and the organic
extract concentrated to 1.5 volumes. Acetic acid (1.38 L) is added and the solution
concentrated to 2 L. Acetic acid (7 L) and zinc powder (2.5 kg, 38.5 mol) are added and
the mixture is heated to 40-50 °C and stirred for 3 hours. EtOAc (9.8L) is added and the
mixture is filtered through diatomaceous earth. The filter cake is washed with EtOAc (4
L). The filtrate is separated and water (7 L) is added to the combined organics.
Ammonium hydroxide is added to reach a pH >9. The layers are separated and the
organic layer is concentrated to 2 L. Ethanol (2.8 L) is added and the solution is
concentrated to 2 L. Ethanol (19 L) is added and the mixture is filtered through
diatomaceous earth to give an ethanol solution of the title compound, which is used
without further purification.
Preparation 5
[(3S,4R)-l-allyl-3-(5-bromo-2-fluoro-phenyl)-4-(hydroxymethyl)pyrrolidin-3-
yl]ammonium;(2S,3S)-4-hydroxy-2,3-bis[(4-methylbenzoyl)oxy]-4-oxo-butanoate
Di-p-toluoyl-L-tartaric acid monohydrate (1.04 kg, 2.69 mol) is added to a
solution of (l-allyl-4-amino-4-(5-bromo-2-fluorophenyl)pyrrolidin-3-yl)metlianol (1264
g. 3.85 mmol) in ethanol (21 L). The mixture is heated to 65-75 °C and stirred for 3
hours. The mixture is cooled to 5-10 °C, a seed crystal is added of [(3S,4R)-l-allyl-3-(5-
bromo-2-fluoro-phenyl)-4-(hydroxymethyl)pyrrolidin-3-yl]ammonium;(2S,3S)-4-
hydroxy-2,3-bis[(4-methylbenzoyl)oxy]-4-oxo-butanoate (1.0 g), and the mixture is
stirred for 3 hours. The solid is filtered and the filter cake is washed with cold ethanol
( 1.4 L). The filter cake is dried to give the title compound as a white solid. Chiral
analysis of the second eluting isomer: Column: IC Chiralpak, 4.6 mm * 250 mm * 5 ;
eluent: 90% hexane (0.3% diethylamine): 10% ethanol (0.3% diethylamine); flow rate of
1.0 mL/min at UV 270 nm confirms the enantiomerically enriched (99% ee) enantiomer
with Rt = 7.4 minutes, (1050 g, 38%). l NMR(400 MHz, CD3OD) 2.40(s, 6H), 3.05-
3.04(m, 1H), 3.57-3.3 l(m, 3H), 3.66-3.58(m, 4H), 3.75-3.74(m, 2H), 5.38-5.36(m, 1H),
5.50-5.46(m, 1H), 5.88(s, 2H), 5.97-5.91(m, 1H), 7.10-7.05(m, 1H), 7.29(d, J=8.0 Hz,
4H), 7.53-7.51(m, 1H), 7.80-7.78(m, 1H), 8.01(d, J=8.0 Hz, 4H).
Preparation 6
((3R,4S)-l-Allyl-4-amino-4-(5-bromo-2-fluorophenyl)pyrrolidin-3-yl)methanol
1N HC1 (500 mL, 500 mmol) is added to a 0 °C solution of [(3S,4R)-l-allyl-3-(5-
bromo-2-fluoro-phenyl)-4-(hydroxymethyl)pyrrolidin-3-yl]ammonium;(2S,3S)-4-
hydroxy-2,3-bis[(4-methylbenzoyl)oxy]-4-oxo-butanoate (100 g, 139.4 mmol) in EtOAc
(500 mL). The mixture is stirred for 1 hour. The aqueous layer is separated and the pH is
adjusted to 8 with 1N NaOH. The aqueous layer is extracted with EtOAc (350 mL 2).
The organic layers are combined, washed with water (500 mL) and concentrated to give
the title compound (40 g, 87%). Chiral analysis of the second eluting isomer: Column: IC
Chiralpak, 4.6 mm * 250 mm * 5 ; eluent: 90% hexane (0.3% diethylamine): 10%
ethanol (0.3% diethylamine); flow rate of 1.0 mL/min at UV 270 nm confirms the
enantiomerically enriched (99.7% ee) enantiomer with Rt = 7.4 minutes. 1H NMR(400
MHz, CDC13) 2.78-2.70(m, 5H), 3.16-3.00(m, 3H), 3.87-3.75(m, 1H), 3.90-3.84(m,
1H), 5.24-5.1 l(m, 2H), 5.91-5.87(m, 1H), 6.95-6.91(m, 1H), 7.35-7.32(m, 1H), 7.67-
7.65 (m, 1H).
Preparation 7
N-(((3S,4R)-l-Allyl-3-(5-bromo-2-fluorophenyl)-4-(hydroxymethyl)pyrrolidin-3-
yl)carbamothioyl)benzamide
Benzoyl isothiocyanate (15.0 g, 91.9 mmol) is added to a 0 °C solution of
((3R,4S)-l-allyl-4-amino-4-(5-bromo-2-fluorophenyl)pyrrolidin-3-yl)methanol (30 g,
91.1 mmol) in THF (400 mL). The solution is warmed to 25 °C and stirred for 1 hour to
give a THF solution of the title compound, which is used without further purification.
Preparation 8
N-((4aR,7aS)-6-Allyl-7a-(5-bromo-2-fluorophenyl)-4,4a,5,6,7,7a-hexahydropyrrolo[3,4-
d][1,3]thiazin-2-yl)benzamide, dihydrochloride
Triphenylphosphine (36.8 g, 140.3 mmol) is added to a THF (400 mL) solution of
N-(((3 S,4R)- 1-allyl-3 -(5-bromo-2-fluorophenyl)-4-(hydroxymethyl)pyrrolidin-3 -
yl)carbamothioyl)benzamide (91.1 mmol). Di-t-butyl azodicarboxylate (31.6 g, 137.2
mmol) in THF (100 mL) is added. The mixture is stirred at 20-30 °C for 2 hours. The
mixture is concentrated and MTBE (400 mL) is added. The solution is filtered through
diatomaceous earth and the cake is washed with MTBE (130 mL). The filtrates are
combined and 1N HC1 in EtOAc (200 mL) is added. The mixture is stirred for 2 hours
and then concentrated to 500 mL. MTBE (320 mL) is added and the solution is filtered
and washed with heptane (130 mL). The solid is slurried in EtOAc (650 mL) and stirred
at 50-60 °C for 2 hours The hot slurry is filtered and the solid is washed with EtOAc
(130 mL) and heptane (130 mL). The solid is reslurried in EtOAc (650 mL) and stirred
for 2 hours at 50-60 °C. The hot slurry is filtered and washed with EtOAc (130 mL) and
heptane (130 mL). The solid is dried to give the title compound as the di-HCl salt (40 g,
80%, 99.5% ee). Chiral analysis of the first eluting isomer: Column: IC Chiralpak, 4.6
mm * 250 mm * 5 ; eluent: 85% hexane (0.1% diethylamine): 15% isopropyl alcohol
(0.1% diethylamine); flow rate of 1.0 mL/min at UV 282 nm confirms the
enantiomerically enriched (99.5% ee) enantiomer with Rt = 12.5 minutes.
Preparation 9
N-((4aR,7aS)-6-Allyl-7a-(2-fluoro-5-(2,2,2-trifluoroacetamido)phenyl)-
4,4a,5 ,6,7,7a-hexahydropyrrolo [3,4-d] [1,3]thiazin-2-yl)benzamide
15% Sodium carbonate (440 mL) is added to a solution of N-((4aR,7aS)-6-allyl-7a-(5-
bromo-2-fluorophenyl)-4,4a,5,6,7,7a-hexahydropyrrolo[3,4-d][l,3]thiazin-2-
yl)benzamide dihydrochloride (495 g, 717.88 mmol) in EtOAc (3 L) and water (784 mL).
The mixture is stirred for 1-2 hours. The layers are separated and the organic layer is
filtered through silica gel (40 g) and washed with EtOAc (600 mL). The filtrate is
concentrated to dryness to give N-((4aR,7aS)-6-allyl-7a-(5-bromo-2-fluorophenyl)-
4,4a,5,6,7,7a-hexahydropyrrolo[3,4-d] [1,3]thiazin-2-yl)benzamide. Trifluoroacetamide
(136.7 g, 1.21 mol), Nal (182.5 g, 1.22 mol), 4 A molecular sieves (342 g), and K2C0 3
(170.9 g, 1.24 mol) are added to a solution of N-((4aR,7aS)-6-allyl-7a-(5-bromo-2-
fluorophenyl)-4,4a,5,6,7,7a-hexahydropyrrolo[3,4-d][l,3]thiazin-2-yl)benzamide (341 g,
494.54 mmol) in DMSO (525 mL) and 1,4-dioxane (1.025 L). Trans-N,N'-
dimethylcyclohexane (81.6 g, 573.66 mmol) and copper iodide (27.3 g, 143.34 mmol) in
DMSO (500 mL) are added to the reaction mixture. The mixture is stirred for 5 minutes.
The mixture is warmed to 100 °C and stirred for 8 hours and cooled to 24 °C. Water (5.9
L) and DCM (5.9 1) are added, the mixture is filtered, and the layers are separated. The
organic layer is washed with water (5.9 L) to obtain the title compound in a solution of
DCM, which is used without further purification.
Preparation 10
N-((4aR,7aS)-6-Allyl-7a-(5-amino-2-fluorophenyl)-4,4a,5,6,7,7a-hexahydropyrrolo[3,4-
d] [1,3]thiazin-2-yl)benzamide, hydrochloride
Sodium hydroxide (28.7 g) and water (2.7 L) are added to a DCM solution of N-
((4aR,7aS)-6-allyl-7a-(2-fluoro-5-(2,2,2-trifluoroacetamido)phenyl)-4,4a,5,6,7,7ahexahydropyrrolo[
3,4-d][l,3]thiazin-2-yl)benzamide (250 g, 494.4 mmol) and the
mixture is stirred at 24 °C for 68 hours. 1N HCI (3.5 L) is added to obtain a pH of 1-3.
The layers are separated and the aqueous layer is washed with DCM (680 mL). DCM (4
L) is added to the aqueous followed by 21% ammonium hydroxide to obtain a pH of 8-
10. The layers are separated and organic extracts are combined, filtered through silica gel
(170 g) and washed with DCM (1.4 L). The solvent is concentrated to dryness and
diluted with EtOAc (4 L). 1N HCI in EtOAc (700 mL) is added at a temperature below
25 °C and the mixture is stirred for 1 hour. The mixture is concentrated to about 7-8
volumes and EtOAc (2.8 L) is added. The resulting precipitate is filtered and washed
with EtOAc (400 mL). The solid is dried to give the title compound. (246 g, 52%).
Preparation 11
N-((4aR,7aS)-7a-(5-Acetamido-2-fluorophenyl)-6-allyl-4,4a,5,6,7,7ahexahydropyrrolo
[3,4-d] [1,3]thiazin-2-yl)benzamide
Acetic anhydride (23.5g, 0.23 mol) is added to a solution of N-((4aR,7aS)-6-allyl-
7a-(5-amino-2-fluorophenyl)-4,4a,5,6,7,7a-hexahydropyrrolo[3,4-d] [1,3]thiazin-2-
yl)benzamide hydrochloride (100 g, 0.153 mol) and triethylamine (54.3 g, 0.535 mol) in
DCM (800 mL). After stirring for 1 hour at 20 - 25 °C, saturated NaHC0 3 (700 mL) and
water (600 mL) are added. The layers are separated to give the title compound, which is
used without further purification as a solution in DCM.
Preparation 12
N-((4aR,7aS)-7a-(5-Acetamido-2-fluorophenyl)-4,4a,5,6,7,7a-hexahydropyrrolo[3,4-
d][1,3]thiazin-2-yl)benzamide
Triphenylphosphine (4.0g, 0.015 mol) and 1,3-dimethylbarbituric acid (15.2 g,
0.097 mol) are added to a DCM solution of N-((4aR,7aS)-7a-(5-acetamido-2-
fluorophenyl)-6-allyl-4,4a,5,6,7,7a-hexahydropyrrolo[3,4-d][l,3]thiazin-2-yl)benzamide
(0.153 mol). Palladium acetate (1.7g, 7.7 mmol) is added and the mixture is stirred at 20
to 30 °C for 1 hour. 25% Ammonium hydroxide is added and the layers are separated.
The organic layer is washed with HOAc (3.0 equiv in 500 mL of water) and the pH is
adjusted to 8-9 with 25% ammonium hydroxide. The aqueous layer is extracted with
DCM (2 500 mL). The organic extracts are combined and concentrated to 3-4 volumes.
MTBE ( 1 L) is added and the mixture is filtered. The mixture is concentrated and
heptane ( 1 L) is added. The resulting solid is filtered, collected, and dried to give the title
compound (48 g, 76%). l NMR (400 MHz, CDC13) 2.15 (s, 3H), 2.87-2.83(m, 1H),
3.43-3.23(m, 5H), 3.70-3.67(m, 1H), 7.12-7.07 (m, 1H), 7.28-7.27 (m, 1H), 7.52-7.41(m,
4H), 7.79(m, 1H), 8.18-8.16(m, 2H). ES m/z 413.1 (M+l).
Preparation 13
N-((4aR,7aS)-7a-(5-Acetamido-2-fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5, 6,7,7ahexahydro
rrolo[3,4-d] [1,3]thiazin-2-yl)benzamide
2-Chloro-5-fluoropyrimidine (28.9 g, 218 mmol) and potassium carbonate (33.46
g, 242.1 mmol) are added to a solution of N-((4aR,7aS)-7a-(5-acetamido-2-fluorophenyl)-
4,4a,5,6,7,7a-hexahydropyrrolo[3,4-d][l,3]thiazin-2-yl)benzamide (50 g, 121.22 mmol)
in DMF (100 mL). The mixture is heated to 80-85 °C for 8 hours. The mixture is cooled
to 24 °C, filtered, and washed with DMF (100 mL). The solids are slurried in water (2 L)
and filtered to obtain the title compound (68.5g, 98%). LC-MS: m/z=509.2 (M+l)+,
NMR (400 MHz, de-DMSO) ppm 1.22 (t, J = 7.28 Hz, 2 H) 1.92 - 2.07 (m, 6 H) 2.89 -
3.20 (m, 2 H) 3.36 - 3.44 (m, 1 H) 3.67 (t, J=9.54 Hz, 1 H) 3.84 (br. s., 1 H) 4.16 (br. s., 2
H) 7.23 (br. s., 2 H) 7.35 - 7.61 (m, 8 H) 7.77 (br. s., 2 H) 7.85 - 8.18 (m, 4 H) 8.48 (s, 4
H) 10.15 (br. s., 1 H) 10.46 - 10.59 (m, 1 H).
Preparation 14
(4aR,7aS)-7a-(5-Amino-2-fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,6,7,7ahexahydropyrrolo
[3,4-d] [1,3]thiazin-2-amine
Lithium hydroxide (8.6 g, 204.9 mmol) is added to a solution of N-((4aR,7aS)-7a-
(5-acetamido-2-fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,6,7,7ahexahydropyrrolo[
3,4-d][l,3]thiazin-2-yl)benzamide (80 g, 157.3 mmol) in methanol
(400 mL). The mixture is heated to 60-70 °C for 4 hours. Concentrated HC1 (132 g) is
added and the mixture is stirred at 55 °C for 18 hours. The mixture is cooled to 30 °C and
concentrated to remove the methanol. Water is added and the aqueous layer is extracted
with DCM (3 x ) to obtain the title compound as an aqueous solution of 920 g of which
5.6% of the total mass is the title compound which is used without further purification.
Preparation 15
Benzyl N-allyl-N-(2,2-dimethoxyethyl)carbamate
A solution of benzyl N-(2,2-dimethoxyethyl)carbamate (50 g, 208.9 mmol) in
toluene (180 mL) is treated with solid potassium hydroxide (51.6 g, 919.69 mmol) under
nitrogen. After 10 minutes, benzyltriethylammonium chloride (0.8 g, 3.1 mmol) is added.
After another 10 minutes a solution of allyl bromide (33 g, 272.8 mmol) in toluene (50
mL) is added drop wise over 10 minutes. The resultant mixture is stirred at 50 °C for 48
hours. The mixture is cooled to room temperature and quenched with water. The organic
layer is separated, washed with brine, dried over magnesium sulfate, and concentrated to
dryness to give the title compound (44 g, 75%). ES/MS (m/e): 280 (M+H).
Preparation 16
Benzyl N-allyl-N-(2-oxoethyl)carbamate
A solution of benzyl N-allyl-N-(2,2-dimethoxyethyl)carbamate (30 g, 107 mmol)
in formic acid (36.8 mL, 860 mmol) and water (4.84 mL) is stirred at room temperature
overnight. The mixture is concentrated and diluted with hexanes/EtOAc (1:2) and water.
The organic layer is separated, washed with brine solution until pH=6, and dried over
sodium sulfate. The solvent is evaporated to give the title compound (25 g, 99%).
ES/MS (m/e): 234 (M+H).
Preparation 17
Benzyl N-allyl-N-[2-hydroxyiminoethyl]carbamate
A solution of benzyl N-allyl-N-(2-oxoethyl)carbamate (25 g, 107 mmol) in
acetonitrile (150 mL) is treated with hydroxylamine hydrochloride (9.68 g, 139 mmol)
and a solution of sodium acetate trihydrate (16 g, 117.9 mmol) in water (75 mL). The
mixture is stirred at room temperature overnight. The acetonitrile is evaporated and the
aqueous solution is extracted with EtOAc. The organic layer is separated, dried over
magnesium sulfate, and concentrated under vacuum to give the title compound (24 g,
90%). ES/MS (m e): 249 (M+H).
Preparation 18
Benzyl 3,3a,4,6-tetrahydropyrrolo [3,4-c]isoxazole-5-carboxylate
A solution of benzyl N-allyl-N-[2-hydroxyiminoethyl]carbamate (24 g, 96.6
mmol) in DCM (338 mL) is treated drop wise over 10 minutes with a 5% w/w aqueous
solution of sodium hypochlorite (106.08 mmol, 143.06 mL). The resultant mixture is
stirred at room temperature overnight. The reaction is quenched with a 40 % aqueous
solution of sodium bisulfite (7 g). The organic layer is separated, dried over magnesium
sulfate, and concentrated under vacuum. The crude product is purified over silica gel
eluting with 5 % EtOAc in hexanes to give the title compound ( 18 g, 75 %). ES/MS
(m/e): 247 (M+H).
Preparation 19
Benzyl 6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydro-lH-pyrrolo[3,4-c]isoxazole-5-
carbox late
A 1.6 M hexanes solution of n-butyl lithium (25.4 mL, 40.6 mmol) is added drop
wise to a -78 °C solution of 4-bromo-l-fluoro-2-iodobenzene (12.22 g, 40.6 mmol) in
THF (60 mL) to give a yellow solution that is stirred at -78 °C for 15 minutes.
Boron trifluoride etherate (5.14 mL, 40.6 mmol) is added to a separate -78 °C solution of
benzyl 3,3a,4,6-tetrahydropyrrolo[3,4-c]isoxazole-5-carboxylate (5 g, 20.3 mmol) in THF
(60 mL) and the mixture is stirred at -78 °C for 5 minutes . This solution is added to the
previously prepared -78 °C organolithium mixture via cannula. The combined mixture is
stirred for 30 minutes at -78 °C. The mixture is quenched with saturated aqueous
ammonium chloride and warmed to room temperature. The mixture is extracted with
EtOAc (3 x ) and the organic extracts are combined, dried over sodium sulfate, filtered
and the solvent removed in vacuo. The crude product is purified over silica gel with a 35
minute 5% to 100% EtOAc in hexanes gradient to give the title compound (2.27 g, 27%).
ES/MS (m e): (7 Br/ 1Br) 421/423 (M+H).
Preparation 20
Benzyl l-(benzoylcarbamothioyl)-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-
tetrahydropyrrolo[3,4-c]isoxazole-5-carboxylate
Benzoyl isothiocyanate (2.87 mL, 21.28 mmol) is added drop wise to a solution of
benzyl 6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetraliydro-lH-pyrrolo[3,4-c]isoxazole-5-
carboxylate (5.977 g, 14.2 mmol) in THF (95 mL) and stirred overnight under nitrogen.
The solvent is removed in vacuo. The crude product is purified over silica gel with a 30
minute 5% to 100% EtOAc in hexanes gradient to give the title compound (6.05 g, 73%).
ES/MS (m/e): (7 Br/ 1Br) 584/586 (M+H).
Preparation 2 1
Benzyl 3-(benzoylcarbamothioylamino)-3 -(5 -bromo-2-fluoro-phenyl)-4-
(hydroxymethyl)pyrrolidine- 1-carboxylate
A mixture of benzyl l-(benzoylcarbamothioyl)-6a-(5-bromo-2-fluoro-phenyl)-
3,3a,4,6-tetrahydropyrrolo[3,4-c]isoxazole-5-carboxylate (6.05 g 10.4 mmol) and zinc
(dust, <10 micron) (6.77 g, 103.5 mmol) is stirred in acetic acid (52 mL) at room
temperature overnight under nitrogen. The reaction is diluted with EtOAc and filtered
through diatomaceous earth. The solvent is removed in vacuo and the residue is diluted
with EtOAc, water, and saturated aqueous sodium bicarbonate. The mixture is extracted
with EtOAc (3 x), the combined organic layers are combined and dried over sodium
sulfate, filtered, and the solvent removed in vacuo. The crude product is purified over
silica gel with a 30 minute 5% to 100% EtOAc in hexanes gradient to give the title
compound (5.222 g, 86%). ES/MS (m e): (7 Br/ 1Br) 586/588 (M+H).
Preparation 22
Benzyl 2-benzarnido-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetraliydropyrrolo[3,4-
d] [1,3]thiazine-6-carboxylate
,-carbonyldiimidazole (2.87 g, 17.7 mmol) is added to a solution of benzyl 3-
(benzoylcarbamothioylamino)-3 -(5 -bromo-2-fluoro-phenyl)-4-
(hydroxymethyl)pyrrolidine-l-carboxylate (5.198 g, 8.86 mmol) in THF (52 mL). The
mixture is stirred for 1.5 hours at room temperature and then the reaction is heated at
reflux overnight under nitrogen. The reaction is cooled, diluted with water, and extracted
with EtOAc (3 x). The organic layers are combined, dried over sodium sulfate, filtered,
and the solvent removed in vacuo. The crude product is purified over silica gel with a 30
minute 5% to 100% EtOAc in hexanes gradient to give the title compound (2.93 g, 58%).
ES/MS (m/e): (7 Br/ 1Br). 568/570 (M+H)
Preparation 23
N-[7a-(5-Bromo-2-fluoro-phenyl)-4a,5,6,7-tetrahydro-4H-pyrrolo[3,4-d][l,3]thiazin-2-
yljbenzamide
Iodotrimethylsilane (2.21 mL, 15.46 mmol) is added drop wise to a room
temperature solution of benzyl 2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazine-6-carboxylate (2.93 g, 5.15 mmol) in acetonitrile
(44 mL). The reaction is stirred at room temperature for two hours and the solvent is
removed in vacuo. The crude product is purified with an SCX column using 3:1
DCM:methanol and then 2:1 DCM:7 N ammonia in methanol to give the title compound
(2.098 g, 94%). ES/MS (m e): (7 Br/ 1Br) 434/436 (M+H).
Preparation 24
t r t-Butyl 2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d] [1,3]thiazine-6-carboxylate
Di-t-butyldicarbonate (1.16 g, 5.31 mmol) and triethylamine (1.01 mL, 7.25
mmol) are added to a solution of N-[7a-(5-bromo-2-fluoro-phenyl)-4a,5,6,7-tetrahydro-
4H-pyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide (2.098 g, 4.83 mmol) in DCM (48 mL).
The reaction is stirred for 1 hour at room temperature under nitrogen. The solvent is
removed in vacuo and the crude product is purified over silica gel with a 30 minute 5% to
100% EtOAc in hexanes gradient to give the title compound (2.556 g, 99%). ES/MS
(m/e): (7 Br/ 1Br) 534/536 (M+H).
Preparation 25
t rt-Butyl 7a-(5-amino-2-fluoro-phenyl)-2-benzamido-4,4a,5,7-tetrahydropyrrolo[3,4-
d] [1,3]thiazine-6-carboxylate
A solution of t r t-butyl 2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazine-6-carboxylate (2.556 g, 4.8 mmol) andtrans-N,N'-
dimethyl-l,2-cyclohexanediamine (150 mg, 1.1 mmol) in ethanol (50 mL) is treated with
sodium azide (933 mg, 14.3 mmol). An aqueous solution of L-ascorbic acid sodium salt
(0.66 M, 3.2 mL, 2.1 mmol) and water ( 1 mL) are added and the top of the flask is purged
with nitrogen. The mixture is treated with an aqueous solution of copper(II)sulfate
pentahydrate (0.33 M, 3.2 mL, 1.1 mmol) and the mixture is immediately heated on a
preheated hot plate at 80 °C for 1.5 hrs under nitrogen. A homogeneous mixture is
obtained upon heating. The reaction is cooled, diluted with ice water, and the mixture is
extracted with EtOAc (3 x). The organic extracts are combined and dried over sodium
sulfate, filtered, and the solvent removed in vacuo to give the crude azide product. The
crude azide product is combined with 10% palladium on carbon ( 1 g) in cold ethanol (150
mL) and the mixture is purged using vacuum/nitrogen and then vacuum/hydrogen. The
mixture is stirred at room temperature under 30 psi of hydrogen for 5 hours. The reaction
is vented, filtered through diatomaceous earth, and the filter cake rinsed with DCM. The
solvent is removed from the filtrate in vacuo and the crude product is purified over silica
gel with 50% EtOAc in DCM to give the title compound (2.014 g, 89%). ES/MS (m/e):
471 (M+H).
Preparation 26
N-[7a-(5-Amino-2-fluoro-phenyl)-4a,5,6,7-tetrahydro-4H-pyrrolo[3,4-d][l,3]thiazin-2-
yljbenzamide
Trifluoroacetic acid (10 mL) is added to a solution of t rt-butyl 7a-(5-amino-2-
fluoro-phenyl)-2-benzamido-4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazine-6-carboxylate
(2.013 g, 4.28 mmol) in DCM (30 mL) and the mixture is stirred at room temperature
under nitrogen for 4 hours. The solvent removed in vacuo and the crude product is
purified with an SCX column using 3:1 DCM:methanol and then 2 :1 DCM:7 N ammonia
in methanol to give the title compound (1.555 g, 98%). ES/MS (m e): 371 (M+H).
Preparation 27
N-[7a-(5-Amino-2-fluoro-phenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d] [1,3]thiazin-2-yl]benzamide
A solution of N-[7a-(5 -amino-2-fluoro-phenyl)-4a,5 ,6,7-tetrahydro-4Hpyrrolo[
3,4-d][l,3]thiazin-2-yl]benzamide (705 mg, 1.90 mmol), 5-fluoro-2-
chloropyrimidine (1.01 g, 7.61 mmol), and DIPEA (1.66 mL, 9.52 mmol) are heated in
1,4-dioxane (20 mL) to reflux for 4 hours under nitrogen. The reaction is cooled, diluted
with water, and extracted with EtOAc (3 x). The organic layers are combined, dried over
sodium sulfate, filtered and the solvent removed in vacuo to give crude product. The
crude product is purified over silica gel with a 25 minute 5% to 100% EtOAc in hexanes
gradient to give the title compound (590 mg, 66%). ES/MS (m/e): 467 (M+H).
Preparation 28
N-[(4aR,7aS)-7a-(5-Amino-2-fluoro-phenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide, (isomer 1)
Racemic N-[7a-(5-amino-2-fluoro-phenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide (1.694 g, 3.63 mmol) is purified by
chiral HPLC (Column: Chiralcel OJ, 8 x 35 cm; eluent: 90 % methanol (0.2 %
dimethylethylamine) and 10% acetonitrile; flow 400 mL/min at UV 280 nm). Analysis of
the first eluting isomer (Column: Chiralcel OJ-H 0.46 x 15 cm; eluent: 10:90
acetonitrile:methanol (with 0.2% dimethylethylamine); flow: 0.6 mL/min at UV 280 nm)
confirms the enantiomerically enriched (99% ee) enantiomer with Rt = 6.70 minutes, (723
mg, 43%). ES/MS (m e): 467 (M+H).
Preparation 29
N-[3-[(4aR,7aS)-2-Benzamido-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
telTahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-metlioxy-pyrazine-2-
carboxamide, (isomer 1)
N-[(4aR,7aS)-7a-(5-Amino-2-fluoro-phenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide, isomer 1 (0.361 g, 0.77 mmol) is
dissolved in a mixture of DCM (4 mL) and DMF (0.5 mL). 5-Methoxypyrazine-2-
carboxylic acid (240 mg, 1.55 mmol), HOBT (210 mg, 1.55 mmol) and EDCI (300 mg,
1.55 mmol) are added to the mixture and the mixture is stirred overnight at room
temperature. The reaction solution is added directly onto a 12 g silica gel loading column
and purified using a 40 g silica gel column and eluting with a 0-100% EtOAc/hexanes
gradient. The product is dissolved in EtOAc (200 mL), washed with 1N NaOH (2 x 50
mL), and with brine ( 1 50 mL). The silica gel purification is repeated as described
above to give the title compound (350 mg, 74%). ES/MS (m/e): 603 (M+H).
Preparation 30
5-Allyl-6a-(2-fluorophenyl)-3,3a,4,6-tetrahydro-lH-pyrrolo[3,4-c]isoxazole
Flow chemistry reaction step: A 343-ml seamless stainless steel tubular reactor
(O.D=l/8 inch) is placed inside a GC oven and flushed with toluene at 20 mL/min for 20
minutes. Apply back pressure of nitrogen (720 psig) and set the temperature of the GC to
210 °C. After the temperature has reached 210 °C, a solution of 2-(diallylamino)-l-(2-
fluorophenyl)ethanone oxime (480.51 g, 1.74 mol) in toluene (5.81 L) is pumped through
the reactor at 22.866 mL/min using a pair of high-pressure syringe pumps working in
continuous mode to give a residence time of 15 minutes. After all the stock solution is
consumed the reactor is flushed with toluene at 22.866 mL/min for 30 minutes. The
temperature of the GC oven is set to 25 °C and the complete solution is collected and
concentrated under vacuum. The solvent is evaporated and residue dissolved in
methylene chloride (2.5 L) and water (5 L). The pH is adjusted to 1 with hydrochloric
acid and the aqueous layer is separated and neutralized with sodium hydroxide to adjust
the pH to 10. The aqueous layer is extracted with MTBE (3 2.5 L). The organic
extracts are combined, dried over sodium sulfate, filtered, and evaporated to dryness to
give the crude title compound (248 g, 47%) which is used without further purification.
ES/MS (m e): 249 (M+l).
Preparation 31
1-Allyl-4-amino-4-(2-fluorophenyl)pyrrolidin-3 -yljmethanol
Zinc dust (590 g, 9 mol) is added to a solution of 5-allyl-6a-(2-fluorophenyl)-
3,3a,4,6-tetrahydro-lH-pyrrolo[3,4-c]isoxazole (3559 g, 1.29 mol) in a mixture of
methanol (2.85 L) and ammonium chloride saturated aqueous solution (3.56 L) and
mixture is heated for 16 hours at 70 °C. The reaction is cooled to 60 °C, diluted with THF
(2.85 L), and filtered while hot over diatomaceous earth. The filtrate is evaporated to
remove the organic solvent, and the aqueous mixture is diluted with citric acid 10% w/w
aqueous solution (4 L) and EtOAc (3.5 L). The organic layer is separated and the
aqueous layer washed with EtOAc ( x 2 L). The aqueous layer is neutralized with
sodium hydroxide 50% w/w to adjust the pH to 10, and then is extracted with EtOAc (2 x
1.5 L). The organic extracts are combined, dried over sodium sulfate, filtered, and
evaporated to dryness to give the crude title compound (299 g, 92%). ES/MS (m/e): 251
(M+l).
Preparation 32
[(3R,4S)-l-Allyl-4-amino-4-(2-fluorophenyl)pyrrolidin-3-yl]methanol; 2,3-bis[(4-
methylbenzoyl)oxy]butanedioic acid
A solution of di-/>-toluoyl-L-tartaric acid (348.6 g, 884 mmol) in 1-methoxy-2-
propanol (1.13 L) is added to a solution of [(3R,4S)-l-allyl-4-amino-4-(2-
fluorophenyl)pyrrolidin-3-yl]methanol (225.9 g, 902 mmol), in 1-methoxy-2-propanol
(1.13 L) previously heated at 40 °C. The reaction is cooled to 22 °C and stirred for 18
hours. A white solid is collected by filtration and washed with l-methoxy-2-propanol
(600 ml). The collected solid is dried to give the title compound (183.01 g, 31.8%).
ES/MS (m/e): 251 (M+l).
Preparation 33
[(3R,4S)-l-Allyl-4-amino-4-(2-fluorophenyl)pyrrolidin-3-yl]methanol
[(3R,4S)-l-Allyl-4-amino-4-(2-fluorophenyl)pyrrolidin-3-yl]methanol;2,3-bis[(4-
methylbenzoyl)oxy]butanedioic acid (211 g, 331 mmol) is dissolved in water (2.1 L) and
EtOAc (2.3 L). Hydrochloric acid 35% w/w is added to adjust the pH to 1. The aqueous
layer is separated and the pH adjusted to 10 with sodium hydroxide 50% w/w and
extracted with EtOAc (2*). The pH of the aqueous layer is adjusted to 10 with aqueous
NaOH, and extracted with MTBE (3x) while also maintaining the pH of the aqueous
solution at pH=10. The organic extracts are combined, dried over sodium sulfate,
filtered, and concentrated to dryness to give the crude title compound, (73 g, 88%, 94.8%
ee). The product is analyzed by chiral chromatography: Column AS-H, eluent 10%
isopropyl alcohol, 2% isopropyl amine; flow rate of 3 mL/min at UV 220; pressure of 100
bar at 35 °C to give the title compound as the second eluting isomer, Rf = 2.26 minutes.
ES/MS (m e): 251 (M+l).
Preparation 34
N-[(4aR,7aS)-6-Allyl-7a-(2-fluorophenyl)-4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-
2-yl]benzamide
A solution of [(3R,4S)-l-allyl-4-amino-4-(2-fluorophenyl)pyrrolidin-3-
yllmethanol (129.7 g, 414 mmol) in THF (2,3L) is cooled at 0 °C under a nitrogen
atmosphere. Benzoyl isothiocyanate (61.5 mL, 456 mmol) is added keeping the
temperature below 5 °C. The reaction is warmed to room temperature over 3 hours and
,-carbonyldiimidazole (87.4 g, 538.9 mmol) is added and the reaction stirred at 22 °C
for 1 hour followed by heating at 70 °C for 16 hours. The reaction mixture is cooled to 22
°C and the solvent is evaporated. The residue is partitioned in EtOAc ( 1 L) and water ( 1
L). The organic layer is separated and the aqueous layer is extracted with EtOAc (2 
400 mL). The organics are combined, dried over sodium sulfate, filtered, and evaporated
to dryness to give the crude title compound. The crude product is purified by silica gel
chromatography eluting with a gradient of EtOAc / DCM from 0-40% DCM to give the
title compound as pale yellow solid (170 g, 99%) containing residual solvent. ES/MS
(m e): 396 (M+l).
Preparation 35
N-[(4aR,7aS)-7a-(2-Fluorophenyl)-4a,5,6,7-tetrahydro-4H-pyrrolo[3,4-
d][1,3]thiazin-2-yl]benzamide
Benzoic Acid, 2-mercapto- (122 g, 793 mmol),
bis(dibenzylideneacetone)palladium (4.15 g, 7.21 mmol), and 1,4-
bis(diphenylphosphino)butane (3.14 g, 7.21 mmol) are added to a solution of N-
[(4aR,7aS)-6-allyl-7a-(2-fluorophenyl)-4,4a,5 ,7-tetrahydropyrrolo[3 ,4-d] [1,3]thiazin-2-
yljbenzamide (178.21 g, 360 mmol) in anhydrous 2-methyltetrahydrofuran (1.96 L) under
a nitrogen atmosphere. The solution is degassed by vacuum / nitrogen cycles three times,
and then nitrogen is bubbled through the reaction for 15 minutes. The reaction mixture is
heated to 40 °C while bubbling nitrogen through the reaction. When reaction reaches 40
°C the bubbling is removed and reaction mixture is stirred at 40 °C for 3 hours under a
nitrogen atmosphere. The reaction is cooled to 22 °C and diluted with water (2 L). HC1
(5 M) solution is added to adjust the pH to 1. The aqueous layer is separated and washed
with additional EtOAc (2 800 mL). The pH of the aqueous layer is adjusted to 10 with
sodium hydroxide 50% w/w and then is extracted with EtOAc (10 L). The aqueous layer
is washed with additional EtOAc (2 x 750 mL). The organic extracts are combined,
washed with brine, dried over sodium sulfate, filtered, and evaporated to dryness to give
the crude title compound as pale yellow solid (124.7 g, 97%). ES/MS (m/e): 356 (M+l).
Preparation 36
N-[(4aR,7aS)-7a-(2-Fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d] [1,3]thiazin-2-yl]benzamide
A solution of N-[(4aR,7aS)-7a-(2-fluorophenyl)-4a,5,6,7-tetrahydro-4Hpyrrolo[
3,4-d][l,3]thiazin-2-yl]benzamide (124.7 g, 256 mmol), DIPEA (67 mL), 5-
fluoro-2-chloropyrimidine (29.3 ml, 307 mmol) in N-methylpyrrolidone (997 mL) is
heated to 100 °C for 16 hours. The reaction is cooled to 22 °C and poured into cooled
water at 10 °C (10 L) keeping temperature below 15 °C. A pale cream solid is collected
by filtration and washed with additional water. The wet solid is dissolved in EtOAc (2 L)
and transferred to a separator funnel. Sodium chloride aqueous solution 5% w/w ( 1 L) is
added and the organic layer is separated, dried over sodium sulfate, filtered, and the
filtrate evaporated under reduced pressure. The product is purified by silica gel
chromatography using a gradient of 0-40% EtOAc/isohexane to give the title compound
as a pale yellow solid ( 116 g, 70%). ES/MS (m/e): 452 (M+l).
Preparation 37
(4aR,7aS)-7a-(2-Fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-2-amine
Lithium hydroxide (9.26 g, 386 mmol) is added to a mixture of N-[(4aR,7aS)-7a-
(2-fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d][l,3]thiazin-2-yl]benzamide (158.6 g, 351.6 mmol), in methanol (1.6 L). The mixture
is heated at 70 °C for 4 hours and then cooled to 22 °C. The reaction mixture is
evaporated under vacuum to a yellow residue. The residue is partitioned in water ( 1 L)
and EtOAc (750 mL). HC1 (5 M aqueous solution) is added to adjust the pH to 1. The
aqueous layer is separated and the organic layer is washed with EtOAc (2 200 mL).
The pH of the aqueous layer is adjusted with sodium hydroxide 50% w/w aqueous
solution to pH=10 and extracted with EtOAc (3 1 L). The organic extracts are
combined, dried over sodium sulfate, filtered, and evaporated under reduced pressure to
give crude title compound as a pale yellow solid (133.3 g, 99%, containing 12% residual
EtOAc). ES/MS (m e): 348 (M+l).
Preparation 38
(4aR,7aS)-7a-(5-A nino-2-fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-2-amine
Sulfuric acid (33.4 ml, 626.6 mmol) is added to a solution of (4aR,7aS)-7a-(2-
fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-d] [1,3]thiazin-
2-amine (45.8 g, 125,3 mmol) in trifluoroacetic acid (626 mL). The mixture is cooled to
0 °C and stirred for 20 minutes. Fuming nitric acid (6.2 mL, 144.1 mmol) is added and
the reaction mixture is warmed to 22 °C and stirred at for 3 hours. The reaction mixture is
evaporated and MTBE is added (250 mL) and evaporated twice. The residue is dried
under vacuum to a constant weight and then is dissolved in water (147 mL) and ethanol
(885 mL) and degassed with bubbling nitrogen for 15 minutes. The solution is
transferred to a pressure reactor and 10% Pd/C paste type 87L (6.6 g, 6.27 mmol) is
added. The mixture is diluted with additional ethanol (700 mL) and pressurized with
hydrogen at 80 psi for 16 hours. The reaction mixture is filtered and then a second
catalyst charge is added of 10% Pd/C paste type 87L (6.6 g, 6.27 mmol) and the mixture
is pressurized to 80 psi and stirred for 3 days in the pressure reactor. The reaction
mixture is purged with nitrogen and filtered over diatomaceous earth. The filtrate is
evaporated and the residue is partitioned between water (200ml) and EtOAc (200 ml).
The aqueous layer is separated, cooled to 5 0 C, and neutralized with ammonium
hydroxide 15% w/w. The aqueous layer is extracted with EtOAc (3 150 mL). The
organics are combined, dried over sodium sulfate, filtered, and evaporated under reduced
pressure to give the title compound as light brown solid (47.7 g, 99% containing residual
EtOAc). ES/MS (m/e): 363 (M+l).
Example 1
N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d][1,3]thiazin-7a-yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-carboxamide hydrochloride.
N-[3-[(4aR,7aS)-2-Benzamido-6<5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-
carboxamide, isomer 1 (0.350 g, 0.58 mmol) is dissolved in THF (2 mL) and then
methanol (4 mL) and ethanol (4 mL) are added. O-Methylhydroxylamine hydrochloride
(495 mg, 5.81 mmol) and pyridine (470 , 5.81 mmol) are added to the mixture and the
reaction is warmed to 50 °C and stirred overnight. Silica gel (-10 g) is added to the
reaction and the mixture is concentrated. The sample, dried onto silica gel, is loaded onto
an empty cartridge and purified eluting with a 0-10% gradient of 7 N ammonia methanol
in DCM. The product is purified a second time on a SCX column using 3:1
DCM:methanol and then 2 :1 DCM:7 N ammonia in methanol. The product is purified a
final time over silica gel with a 0% to 10% gradient of 7 N ammonia methanol in DCM to
give the free base of the title compound. This material is dissolved in DCM (5 mL) and 1
M hydrogen chloride in diethyl ether (0.20 mL, 660 ΐ) is added. The solvent is
removed in vacuo to give the title compound (71 mg, 23%). ES/MS (m/e): 498 (M+H).
X-Rav 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and a scan rate of 0.5 seconds/step, and 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 form, the
relative intensities of the diffraction peaks may vary due to preferred orientation resulting
from factors such as crystal morphology and habit. Where the effects of preferred
orientation are present, peak intensities are altered, but the characteristic peak positions of
the polymorph are unchanged. See, e.g. , The United States Pharmacopeia #23, National
Formulary #18, 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 crystal 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, are adjusted based on NIST 675 standard peaks at
8.853 and 26.774 degrees 2-theta.
Example l a
Crystalline Form 1 N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-methoxy-pyrazine-2-
carboxamide
N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-methoxy-pyrazine-2-
carboxamide (201 mg, 403.20 ΐ ) is mixed with 4 mL of butyl acetate and stirred on
a 120°C stirplate. The solid dissolves after about 5 minutes, giving a clear colorless
solution. The sample is then cooled to room temperature and a white solid precipitates
out of solution. The sample is then slurried at room temperature for 10 minutes, giving a
thick slurry of white solid. The white solid is isolated by vacuum filtration and dried
under air stream for 5 minutes. The resulting cake of white solid is placed in a tared vial
in the 60°C vacuum oven over the weekend to provide the title compound ( 110 mg).
Crystalline Form 1 N-[3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-
carboxamide is the stable crystal form at room temperature and relative humidity less
than about 15%.
Example l b
Crystalline Form 2 N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d] [l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-methoxy-pyrazine-2-
carboxamide (hydrated)
Oxalyl chloride (888 x , 10.2 mmol) is added to a solution of 5-
methoxypyrazine-2-carboxylic acid ( 1.6 g, 10.2 mmol) in acetonitrile (53 mL) and DMF
(848 x ) . After 15 minutes, the freshly prepared solution is added to a solution of
(4aR,7aS)-7a-(5-amino-2-fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d] [l,3]thiazin-2-amine (2.65 g, 7.3 mmol) in a mixture of water (53
mL) and ethanol (53 mL) previously heated at 50 °C. The reaction mixture is stirred at 50
°C for one hour, cooled to 22 °C, and then stirred overnight at this temperature. The
organic solvent is evaporated under vacuum and the aqueous mixture is treated with
sodium hydroxide 50% w/w aqueous solution to adjust pH= 10. A pale cream solid is
isolated and washed with additional water. The solid is diluted with isopropyl alcohol
(2x) and the solvent is evaporated. The crude material is purified by silica gel
chromatography using a gradient of ammoniated methanol (2 N) / methylene chloride.
The fractions containing the desired product are combined and solvent is evaporated to
give the title compound as an off white solid ( 1.9 g, 52%). ES/MS (m/e): 499 (M+l).
Alternative Preparation of Crystalline Form 2 N-[3-[(4aRJaS)-2-Amino-6-(5-
fluoropyrimidin-2-yl)-4,4a,5,7-tetrahvdropyrrolo [3,4-d] [1,31thiazin-7a-yll -4-fluorophenyl]-
5-methoxy-pyrazine-2-carboxamide (hydrated)
Acetonitrile (500 mL) is added to DMF ( 19.2 mL, 248.9 mmol). Oxalyl Chloride
(39.3 g, 309.63 mmol) followed by 5-methoxypyrazine-2-carboxylic acid (46.0 g, 298.4
mmol) is added to the DMF/acetonitrile solution. In a separate flask, the aqueous solution
of (4aR,7aS)-7a-(5-amino-2-fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d] [l ,3]thiazin-2-amine (56.8 g, 156.75 mmol) is added to
acetonitrile (500 mL) and the pH is adjusted to 9 with ammonium hydroxide (95 mL).
This mixture is then heated to 50 -55 °C. The oxalyl chloride solution is added drop wise
and the mixture is stirred for 3 hours. The pH is adjusted to 8-9 with ammonium
hydroxide. The resulting precipitate is filtered, washed with water, and dried to obtain
the title compound (123 g). The solid is slurried in acetone (250 mL) for 1.5 hours and
filtered. The wet cake is washed with acetone to obtain the title compound ( 110 g with
90.5% purity by HPLC). THF ( 1 L) and activated carbon (9 g) are added to the solid and
the mixture is heated to reflux overnight. The mixture is filtered through diatomaceous
earth and washed with THF (150 mL). The organic solution is concentrated to 10
volumes and heated to 60 °C. Water (430 mL) is added and the mixture is stirred at
60 °C for 8 hours. The mixture is cooled to room temperature and stirred for 10 hours.
The resulting solid is filtered, washed with THF/water (7:6) and dried to give the title
compound (69 g, 88%) LC-MS: m/z=499 (M+l), purity: 98.3%. l NMR (400 MHz,
DMSO-i/6) ppm2.99 - 3.07 (m, 2 H) 3.07 - 3.14 (m, 1H) 3.58 - 3.67 (m, 1H) 3.68 -
3.76 (m, 1H) 3.76 - 3.84 (m, 1 H) 4.02 (s, 3 H) 4.07 (d, J=10.92 Hz, 1H) 6.08 (s, 2 H)
7.19 (dd, J=11.98, 8.72 Hz, 1 H) 7.78 - 7.89 (m, 2 H) 8.41 (s, 1 H) 8.44 (s, 2 H) 8.88 (s, 1
H) 10.60 (s, 1 H).
General procedure for Preparation of Crystalline Form 2 N-[3-[(4aR,7aS)-2-Amino-6-(5-
fluoropyrimidin-2-yl)-4,4a,5 J-tetrahvdropyrrolo[3,4-dl[l,31thiazin-7a-yll-4-fluorophenyll-
5-methoxy-pyrazine-2-carboxamide (hydrated)
Slurry N-[3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-
carboxamide in THF at about 23 °C at a concentration of about 7 1 mg/mL solvent. Heat
the slurry with stirring to dissolution which occurs at about 60 °C to about 63 °C. Add
water to the hot solution to provide a THF:water solvent ratio of about 95:5. Seed
crystals of Form 2 N-[3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-
carboxamide are added (about 3 weight % load). The resulting thin slurry is held at about
60 °C to about 63 °C for about 20 minutes, followed by addition of about 5.3 to about 5.5
volumes of water over about 2 to about 4 hours resulting in a THF:water solvent ratio of
about 69:31. The slurry is then held at about 60 °C to about 63 °C for about 30 minutes
and then cooled to about 23 °C over about 1 hour, and then stirred for about 8-12 hours.
The slurry is then filtered, rinsed lightly with THF:water (35:65), and dried for about 8-12
hours under reduced vacuum at about 40 °C to provide the desired crystalline Form 2 N-
[3-[(4aR,7aS)-2-aniino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d][l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-methoxy-pyrazine-2-carboxamide which is
hydrated.
A prepared sample of crystalline Form 2 N-[3-[(4aR,7aS)-2-amino-6-(5-
fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluorophenyl]-
5-methoxy-pyrazine-2-carboxamide is characterized by an XRD pattern using
CuKa radiation as having diffraction peaks (2-theta values) as described in Table 1 below.
Specifically, the pattern contains a peak at 11.8° in combination with one or more of the
peaks selected from the group consisting of 18.6°, 19.3°, and 26.7°; with a tolerance for
the diffraction angles of 0.2 degrees.
Table 1 : X-ray powder diffraction peaks of crystalline Form 2 of Example lb.
Crystalline Form 2 N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-
4,4a,5 ,7-tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl] -5-methoxypyrazine-
2-carboxamide is the stable crystal form at room temperature and relative
humidity greater than about 15%.
Example l c
Crystalline Form 3 N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-
4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-metlioxypyrazine-
2-carboxamide.
Load a thermogravimetric analysis pan with N-[3-[(4aR,7aS)-2-amino-6-(5 -
fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluorophenyl]-
5-methoxy-pyrazine-2-carboxamide and heat to about 170 °C and hold at 170 °C
for about 5 minutes. Cool to room temperature to provide the title compound.
Alternative Preparation of Crystalline Form 3 N-[3-[(4aR,7aS)-2-Amino-6-(5-
fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl] -4-fluorophenyl]
-5-methoxy-pyrazine-2-carboxamide.
Combine N-[3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl] -4-fluoro-phenyl] -5-methoxy-pyrazine-2-
carboxamide (121 mg) with acetonitrile (5 mL) in a vial, and heat on a 90 °C stirplate.
After about 30 minutes, most of the solid dissolves, providing a cloudy solution. Form 3
seeds are added and the sample is stirred for about 1 hour at about 90 °C. Heating is
removed and the mixture is stirred to provide a bright white solid. The solid is isolated by
vacuum filtration, dry under an air stream for about 10 minutes, and then under reduced
vacuum at about 80 °C for about 8 to 12 hours to provide the title compound.
A prepared sample of crystalline Form 3 N-[3-[(4aR,7aS)-2-amino-6-(5-
fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluorophenyl]
-5-methoxy-pyrazine-2 -carboxamide 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 15.7° in combination with one or more of the
peaks selected from the group consisting of 18.1°, 27.0°, and 19.7°; with a tolerance for
the diffraction angles of 0.2 degrees.
Table 2 : X-ray powder diffraction peaks of crystalline Form 3 of Example lc.
In vitro Assay Procedures:
For in vitro enzymatic and cellular assays, test compounds are 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 curve with final compound concentrations ranging from 10
mM 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 human BACE1
Human BACE1 (accession number: AF190725) is 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 IgGi (Fc) polypeptide ( Vasser, et a ,
Science, 286, 735-741 (1999)). This fusion protein of BACE1(1-460) and human Fc,
named BACEl :Fc, is constructed into the pJB02 vector. Human BACEl(l-460):Fc
(½BACEl:Fc) is transiently expressed in HEK293 cells. 250 g cDNA of each
construct is mixed with Fugene 6 and added to 1 liter HEK293 cells. Four days after the
transfection, conditioned media are harvested for purification.
Purification of ½BACEl:Fc.
huBACEl:Fc is purified by Protein A chromatography. The enzyme is stored at - 80 °C
in small aliquots.
BACE1 FRET Assay
Serial dilutions of test compounds are prepared as described above. Compounds
are further diluted 20in KH2P0 4 buffer. Ten L 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 iL of 50 mM KH2P0 4, pH 4.6, 1mM TRITON® X-100, 1 mg/mL Bovine
Serum Albumin, and 15 of FRET substrate) (See Yang, et. ah, J. Neurochemistry,
91(6) 1249-59 (2004)). The content is mixed well on a plate shaker for 10 minutes.
Fifteen _, of two hundred pM human BACEl(l-460):Fc (See Vasser, et ah, Science,
286, 735-741 (1999)) in the KH2P0 4 buffer is added to the plate containing substrate and
test compounds 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 temperature for 16 to 24 h. 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 BACE1
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
EC50 and IC50 values. (See Sinha, et al, Nature, 402, 537-540 (2000)).
The compound of Example 1 is tested essentially as described above and exhibited
a BACE1 IC 0 of 0.615 nM (± 0.101, n=5) [Mean + SEM; SEM = standard error of the
mean]. This data demonstrates that the compound of Example 1 inhibits purified
recombinant BACE1 enzyme activity in vitro.
PDAPP Primary Neuronal Assay
A confirmatory whole cell assay is also run in primary neuronal cultures generated
from PDAPP transgenic embryonic mice. Primary cortical neurons are prepared from
Embryonic Day 16 PDAPP embryos and cultured in 96 well plates (15 x 104 cells/well in
DMEM/F 12 (1:1) plus 10% FBS). After 2 days in vitro, culture media is replaced with
serum free DMEM/F12 (1:1) containing B27 supplement and 2 (final) of Ara-C
(Sigma, CI 768). At day 5 in vitro, neurons are incubated at 37 °C for 24 h in the
presence/absence of inhibitors (diluted in DMSO) at the desired concentration. At the
end of the incubation, conditioned media are analyzed for evidence of beta-secretase
activity, for example, by analysis of Abeta peptides. Total Abeta peptides (Abeta 1-x) are
measured by a sandwich ELISA, using monoclonal 266 as a capture antibody and
biotinylated 3D6 as reporting antibody. Alternatively, Abeta 1-40 and Abeta 1-42
peptides are measured by a sandwich ELISA, using monoclonal 2G3 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.
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 four-parameter logistic equation to obtain
the EC 0 and IC50 values for the Abeta-lowering effect. The compound of Example 1 is
tested essentially as described above and exhibited the following activity for Abeta
lowering effect:
Table 3
Mean + SEM; SEM = standard error of the mean
These data demonstrate that the compound of Example 1 inhibits Abeta
production in whole cells.
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 knockout
animals. For example, the PDAPP mouse model, prepared as described in Games et al.,
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 to 12 month old PDAPP mice, gene knockout mice
or non-transgenic animals are administered compound formulated in vehicles, such as
corn oil, cyclodextran, phosphate buffers, PHARJVIASOLVE®, or other suitable vehicles.
One to twenty-four hours following the administration of compound, animals are
sacrificed, and brains as well as cerebrospinal fluid and plasma are removed for analysis
of Abetas, C99, and sAPP fragments. (See May, et ah, Journal ofNeuroscience, 3 1,
16507-16516 (2011)).
For standard in vivo pharmacology studies, animals are dosed with various
concentrations of compound and compared to a vehicle-treated control group dosed at the
same time. For some time course studies, brain tissue, plasma, or cerebrospinal fluid is
obtained from selected animals, beginning at time 0 to establish a baseline. Compound or
appropriate vehicle is administered to other groups and sacrificed at various times after
dosing. Brain tissue, plasma, or cerebrospinal fluid is obtained from selected animals and
analyzed for the presence of APP cleavage products, including Abeta peptides, sAPPbeta,
and other APP fragments, for example, by specific sandwich ELISA assays. At the end
of the test period, animals are sacrificed and brain tissues, plasma, or cerebrospinal fluid
are analyzed for the presence of Abeta peptides, C99, and sAPPbeta, as appropriate.
Brain tissues of APP transgenic animals may also be analyzed for the amount of betaamyloid
plaques following compound treatment. "Abeta 1-x peptide" as used herein
refers to the sum of Abeta species that begin with residue 1 and ending with a C-terminus
greater than residue 28. This detects the majority of Abeta species and is often called
"total Abeta".
Animals (PDAPP or other APP transgenic or non-transgenic mice) administered
an inhibitory compound may demonstrate the reduction of Abeta or sAPPbeta in brain
tissues, plasma or cerebrospinal fluids and decrease of beta amyloid plaques in brain
tissue, as compared with vehicle-treated controls or time zero controls. For Example 1,
three hours after administration of 0.3, 1, or 3 mg/kg oral dose of the compound, Abeta 1-
x peptide levels are reduced approximately 31%, 39%, and 61% in brain hippocampus,
and approximately 28%, 42%, and 64% in brain cortex, respectively compared to vehicletreated
mice.
Given the activity of Example 1 against BACE enzyme in vitro, these Abeta
lowering effects are consistent with BACE inhibition in vivo, and further demonstrate
CNS penetration of N-[3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-methoxy-pyrazine-2-
carboxamide.
These studies show that compounds of the invention inhibit BACE and are,
therefore, useful in reducing Abeta levels.
WE CLAIM:
1. A compound of formula:
which is crystalline.
2. The crystalline compound according to claim 1 which is Form 2 N-[3-
[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl] -4-fluoro-phenyl] -5-methoxypyrazine-
2-carboxamide.
3. The crystalline compound according to either claim 1 or claim 2 which is
characterized by a substantial peak in the X-ray diffraction spectrum, at
diffraction angle 2-theta of 11.8°, in combination with one or more of the
peaks selected from the group consisting of 18.6°, 19.3°, and 26.7°; with a
tolerance for the diffraction angles of 0.2 degrees.
4. The crystalline compound according to claim 1 which is Form 1 N-[3-
[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl] -4-fluoro-phenyl] -5-methoxypyrazine-
2-carboxamide.
5. The crystalline compound according to claim 1 which is Form 3 N-[3-
[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl] -4-fluoro-phenyl] -5-methoxypyrazine-
2-carboxamide.
6. The crystalline compound according to either claim 1 or claim 5 which is
characterized by a substantial peak in the X-ray diffraction spectrum, at
diffraction angle 2-theta of 15.7° in combination with one or more of the peaks
selected from the group consisting of 18.1°, 27.0°, and 19.7°; with a tolerance
for the diffraction angles of 0.2 degrees.
7. 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 to 6.
8. A method of preventing 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 to
6.
9. A compound according to any one of claims 1 to 6 for use in therapy.
10. A compound according to any one of claims 1 to 6 for use in the treatment of
Alzheimer's disease.
11.A compound according to any one of claims 1 to 6 for use in preventing the
progression of mild cognitive impairment to Alzheimer's disease.
12. A pharmaceutical composition, comprising a compound according to any one
of claims 1 to 6 with one or more pharmaceutically acceptable carriers,
diluents, or excipients.