The present invention relates to a toluenesulfonic acid (tosylate) salt 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, to
5 pharmaceutical compositions comprising the tosylate salt, to methods of using the
tosylate salt 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
10 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.
15 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
20 reduction in plaque burden and synaptic deficits, thus providing significant therapeutic
benefits, particularly in the treatment of Alzheimer's disease.
United States Patent No. 8,841,293 discloses certain BACE inhibitors for
treatment of Abeta peptide-mediated disorders, such as Alzheimer's disease, and in
particular, discloses for example the BACE inhibitor N-[3-[(4aR,7aS)-2-amino-6-(5-
25 fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluorophenyl]-
5-methoxy-pyrazine-2-carboxamide and the corresponding hydrochloride salt.
Novel forms 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 are desired which provide for improved solid state stability and chemical
30 stability for enhanced utilization in the preparation and manufacture of pharmaceutical
formulations. Accordingly, the present invention provides a tosylate salt 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. In addition,
the present invention provides the tosylate salt 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-fluorophenyl]-
5-methoxy-pyrazine-2-carboxamide which is crystalline. The present invention
further provides the tosylate salt 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 5.0° in combination with one or more
of the peaks selected from the group consisting of 19.6°, 13.8°, and 18.5°; with a
tolerance for the diffraction angles of 0.2 degrees.
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 tosylate salt 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. The present invention further provides 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 tosylate salt
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. 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 tosylate salt
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. 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 a tosylate salt 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. 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 tosylate salt 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-fluorophenyl]-
5-methoxy-pyrazine-2-carboxamide.
Furthermore, this invention provides a tosylate salt 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-fluorophenyl]-
5-methoxy-pyrazine-2-carboxamide 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 tosylate salt 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 for the manufacture of a medicament for the treatment of Alzheimer' s
disease or for the treatment of the progression of mild cognitive impairment to
Alzheimer's disease.
In addition, the invention 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 tosylate salt 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 5.0° in combination with one or more
of the peaks selected from the group consisting of 19.6°, 13.8°, and 18.5°; with a
tolerance for the diffraction angles of 0.2 degrees. The invention also provides a tosylate
salt 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 which is characterized by a substantial peak in the X-ray diffraction
spectrum, at diffraction angle 2-theta of 5.0° in combination with one or more of the
peaks selected from the group consisting of 19.6°, 13.8°, and 18.5°; with a tolerance for
the diffraction angles of 0.2 degrees 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. Furthermore, the invention provides a tosylate salt 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 which is
characterized by a substantial peak in the X-ray diffraction spectrum, at diffraction angle
2-theta of 5.0° in combination with one or more of the peaks selected from the group
consisting of 19.6°, 13.8°, and 18.5°; with a tolerance for the diffraction angles of 0.2
degrees, for the manufacture of a medicament for the treatment of Alzheimer's disease or
for the treatment of the progression of mild cognitive impairment to Alzheimer's disease.
The invention further provides a pharmaceutical composition, comprising the
tosylate salt 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 with one or more pharmaceutically acceptable carriers, diluents, or
excipients. This invention also encompasses novel intermediates and processes for the
synthesis of the tosylate salt 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.
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, et al., Arch.
Neurol, 56, 303-308 (1999)). The term "treatment 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 being
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 tosylate salt 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 is 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 to the pharmaceutical compositions, the treatment
methods, and to the new compounds of the invention.
Crystalline tosylate salt 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 which is characterized by a substantial peak in the X-ray diffraction
spectrum, at diffraction angle 2-theta of 5.0° in combination with one or more of the
peaks selected from the group consisting of 19.6°, 13.8°, and 18.5°; with a tolerance for
the diffraction angles of 0.2 degrees is particularly preferred.
One of ordinary skill in the art will appreciate that 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 can exist in tautomeric forms, as depicted in
Scheme A. When any reference in this application to one of the specific tautomers 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 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: "acetonitrile" refers to ACN; "APP"
refers to amyloid precursor protein; "CSF" refers to cerebrospinal fluid; "DCM" refers to
dichloromethane; "DIPEA" refers to diisopropylethylamine, N-ethyl-N-isopropyl-propan-
2-amine, or ,-diisopropylethylamine; "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-hydroxy lbenzotriazole 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 i3 (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 1 N
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%). H NMR(400 MHz, CDC1 ) 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 4
Relative-(3aR,6aS)-5-Benzyl-6a-(5-bromo-2-fluoro-phenyl)-l-[(4-
methoxyphenyl)methyl]-3,3a,4,6-tetrahydropyrrolo[3,4-c]isoxazole, oxalic acid
N-[(4-Methoxyphenyl)methyl]hydroxylamine (15.5 g, 101 mmol) is added to the
solution of 2-[allyl(benzyl)amino]-l-(5-bromo-2-fluoro-phenyl)ethanone at 40 °C,
followed by titanium (IV) isopropoxide (50.3 mL, 169 mmol). The mixture is
deoxygenated by sparging nitrogen through the system for 5 minutes. The mixture is
heated to 90 °C with stirring for 2 hours. The reaction mixture is washed with a solution
of citric acid (36 g, 186 mmol) in water (36 g) and stirred for 30 minutes at room
temperature. Sodium carbonate (100 g, 939 mmol) is added as a saturated solution in
water and the pH is adjusted to approximately 9. The mixture is diluted with water and
toluene as needed to separate the layers. The toluene layer is washed with a solution of
HC1 in water (5.1 mL of 5 N HC1) diluted with water (200 mL) followed by water (200
mL). The toluene layer is concentrated to a thick oil. The oil is dissolved in EtOAc (375
mL) and a solution of oxalic acid (15.4 g, 169 mmol) dissolved in EtOAc (375 mL) is
added dropwise with stirring at room temperature. The mixture is stirred at room
temperature for 2 days, filtered, and the product cake is washed with EtOAc (250 mL).
The solid is dried overnight in vacuo at 40 °C to give the title compound as a cis racemic
mixture (41.7 g, 84%). ES/MS (m z): 498 (M+H).
Preparation 5
Relative N-[3-[(3aR,6aS)-5-Benzyl-l-[(4-methoxyphenyl)methyl]-3,3a,4,6-
tetrahydropyrrolo[3,4-c]isoxazol-6a-yl]-4-fluoro-phenyl]-2,2,2-trifluoro-acetamide
Relative-(3aR,6aS)-5-benzyl-6a-(5-bromo-2-fluoro-phenyl)-l-[(4-
methoxyphenyl)methyl]-3,3a,4,6-tetrahydropyrrolo[3,4-c]isoxazole, oxalic acid (23.6 g,
40.2 mmol) is added to a mixture of DCM (400 mL) and 5 N NaOH aqueous solution
(200 mL). The mixture is stirred for 10 minutes and then the layers are separated. The
organic layer is concentrated to give relative-(3aR,6aS)-5-benzyl-6a-(5-bromo-2-fluorophenyl)-
l-[(4-methoxyphenyl)methyl]-3,3a,4,6-tetrahydropyrrolo[3,4-c]isoxazole (20.0 g,
40.2 mmol). This material is added to dimethyl sulfoxide (360 mL), 2,2,2-
trifluoroacetamide (7.8 g, 68 mmol), sodium iodide (10.3 g, 68.6 mmol), potassium
carbonate (9.54 g, 68.3 mmol), (1R,2R)-N,N' -dimethyl- 1,2-cyclohexanediamine (4.7 g,
32 mmol), and copper (I) iodide (1.53 g, 8.03 mmol). The mixture is deoxygenated by
sparging nitrogen through the system for 10 minutes. The mixture is heated to 1 5 °C,
stirred for 5.5 hours, and cooled to room temperature. EtOAc (250 mL) and water (200
mL) are added and the resultant mixture is stirred for 10-15 minutes. The layers are
separated and the aqueous layer is back extracted with EtOAc (200 mL). The combined
organic layers are washed with ammonium chloride solution 20 wt% aqueous (100 mL)
followed by lithium chloride solution 20 wt% aqueous (100 mL) and water (100 mL).
The organic layer is dried over magnesium sulfate, filtered, and concentrated to dryness
to give the crude title compound as a racemic cis mixture (23.6 g, 111%). ES/MS (m/z):
530 (M+H).
Preparation 6
Relative-3-[(3aR,6aS)-5-Benzyl-l-(4-methoxyphenyl)methyl]-3,3a,4,6-
tetrahydropyrrolo[3,4-c]isoxazol-6a-yl]-4-fluoro-aniline
Relative N-[3-[(3aR,6aS)-5-benzyl-l-[(4-methoxyphenyl)methyl]-3,3a,4,6-
tetrahydropyrrolo[3,4-c]isoxazol-6a-yl]-4-fluoro-phenyl]-2,2,2-trifluoro-acetamide (21.0
g, 39.7 mmol) is added to methanol (250 mL), 5 N aqueous NaOH (105 mL), and ethanol
(105 mL) and the resultant mixture is heated to 50 °C for 2 hours. The reaction mixture is
concentrated under reduced pressure at 35-40 °C to remove methanol and ethanol. The
material is dissolved in a mixture of MTBE (320 mL) and water (300 mL). The layers are
separated and the organic layer is washed with water (100 mL) followed by a mixture of
water (400 mL) and 12 N aqueous HC1 (3.5 mL). The aqueous layer is added to a
mixture of MTBE (400 mL), water (100 mL), and 5 N aqueous NaOH (25 mL) and the
layers are separated. The aqueous layer is washed with MTBE (100 mL). The organic
layers are combined, dried over magnesium sulfate, filtered, and concentrated in vacuo to
give the title compound as a racemic cis mixture (9.8 g, 57%). ES/MS (m z): 435 (M+H).
Preparation 7
Relative-N-3-[(3aR,6aS)-5-Benzyl-l-[(4-methoxyphenyl)methyl]-3,3a,4,6-
tetrahydropyrrolo[3,4-c]isoxazol-6a-yl]-4-fluoro-phenyl]acetamide
Relative-3-[(3aR,6aS)-5-benzyl-l-(4-methoxyphenyl)methyl]-3,3a,4,6-
tetrahydropyrrolo[3,4-c]isoxazol-6a-yl]-4-fluoro-aniline (9.4 g, 22 mmol) is added to
DCM (110 niL), 4-dimettiylaminopyridine (0.08 g, 0.65 mmol), and triethylamine (6.1
mL, 43 mmol) and the reaction is cooled to 0 °C. Acetic anhydride (3.4 g, 33 mmol) is
added to the solution dropwise. The reaction is warmed to room temperature and stirred
for 2 hours. Water (140 mL) is added to the reaction and the mixture is stirred at room
temperature for 10 minutes. The layers are separated and the aqueous layer is washed
with DCM (100 mL). The combined organic layers are dried over magnesium sulfate,
filtered, and concentrated to give the title compound as a racemic cis mixture (10.1 g,
100%). ES/MS (m/z): 477 (M+H).
Preparation 8
5-AUyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydro-lH-pyrrolo[3,4-c]isoxazole
hydrochloride
TFA (4 L, 52.9 mol) is added dropwise to a solution of 5-allyl-6a-(5-bromo-2-
fluoro-phenyl)-l-[(4-methoxyphenyl)methyl-3,3a,4,6-tetrahydropyrrolo[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/MS (m/z): 327.1 (M+H)
Preparation 9
[l-Allyl-4-amino-4-(5-bromo-2-fluoro-phenyl)pyrrolidin-3-yl]methanol
A saturated aqueous solution of sodium carbonate is added to a solution of 5-allyl-
6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydro-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. HOAc (1.38 L) is added
and the solution concentrated to 2 L. HOAc (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 10
[(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-fluoro-phenyl)pyrrolidin-3-yl]niethanol (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 R, = 7.4 minutes, (1050 g, 38%). H 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 11
Relative-l-[3-[(3S,4R)-3-Amino-4-(hydroxymethyl)pyrrolidin-3-yl]-4-fluorophenyl]
propan-2-one, di-toluenesulfonic acid
Relative-N-3-[(3aR,6aS)-5-benzyl-l-[(4-methoxyphenyl)methyl]-3,3a,4,6-
tetrahydropyrrolo[3,4-c]isoxazol-6a-yl]-4-fluoro-phenyl]acetamide (5.0 g, 11.0 mmol) is
added to water (75 mL), 10% palladium on carbon (2.5 g, 23 mmol), p-toluenesulfonic
acid (5.0 g, 26 mmol), and ethanol (75 mL) and the mixture is hydrogenated under 50 psi
hydrogen atmosphere at room temperature for 20 hours. The mixture is filtered through
diatomaceous earth and the pad is washed with ethanol (100 mL) followed by DCM (100
mL). The filtrate is concentrated to give the title compound as a racemic cis mixture in
quantitate crude yield (8.1 g, >100%). The material is used directly without further
purification. ES/MS (m/z): 268 (M+H).
Preparation 12
((3R,4S)-l-Allyl-4-amino-4-(5-bromo-2-fluoro-phenyl)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 x 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. 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 13
Relative-N-[3-[(3S,4R)-3-Amino-l-(5-fluoropyrimidin-2-yl)-4-
(hydroxymethyl)pyrrolidin-3-yl]-4-fluoro-phenyl]acetamide
Relative-l-[3-[(3S,4R)-3-Amino-4-(hydroxymethyl)pyrrolidin-3-yl]-4-fluorophenyl]
propan-2-one, di toluenesulfonic acid (7.5 grams, 12.3 mmol) is added to ACN
(75 mL). 2-Chloro-5-fluoropyrimidine (2.44 g, 18.4 mmol) and DIPEA (7.5 mL, 43
mmol) are added and the mixture is heated to 70 °C for 20 hours. The mixture is cooled
to room temperature and concentrated. Water (50 mL) is added the mixture is stirred for
1 hour as a slurry forms. The mixture is filtered and dried at room temperature under
vacuum to give the title compound as a racemic cis mixture (3.0 g). The remaining
aqueous solution is extracted with chloroform (3 x 20 mL), dried over sodium sulfate,
concentrated to dryness and dried under vacuum to give additional title compound (1.8 g),
for a total crude yield (4.8 g, 110%). ES/MS (m/z): 364 (M+H).
Preparation 14
N-(((3S,4R)-l-Allyl-3-(5-bromo-2-fluoro-phenyl)-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-fluoro-phenyl)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 15
N-((4aR,7aS)-6-Allyl-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d][l,3]thiazin-2-yl)benzamide, dihydrochloride
Triphenylphosphine (36.8 g, 140.3 mmol) is added to a THF (400 mL) solution of
N-(((3S,4R)-l-allyl-3-(5-bromo-2-fluoro-phenyl)-4-(hydroxymethyl)pyrrolidin-3-
yl)carbamothioyl)benzamide (91.1 mmol). Di-i-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 16
N-[(4aR,7aS)-6-Allyl-7a-[2-fluoro-5-[(2,2,2-trifluoroacetyl)amino]phenyl]-4,4a,5,7-
tetrahydropyrrolo[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-fluoro-phenyl)-4,4a,5,7-tetrahydropyrrolo[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-fluoro-phenyl)-
4,4a,5,7-tetrahydropyrrolo[3,4-d][l,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-fluoro-phenyl)-
4,4a,5,7-tetrahydropyrrolo[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 17
Relative-N-[(4aRJaS)-7a-5-(Acetamido)-2-fluoro-phenyl)-6-(5-fluoropyrimidin-2-yl)-
4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide
THF (30 mL) is added to relative-N-[3-[(3S,4R)-3-amino-l-(5-fluoropyrimidin-2-
yl)-4-(hydroxymethyl)pyrrolidin-3-yl]-4-fluoro-phenyl]acetamide (4.1 g, 11.0 mmol).
The mixture is cooled in an ice bath to 0 °C and benzoyl isothiocyanate (1.50 mL, 11.0
mmol) is added. The mixture is then allowed to warm room temperature over 30 minutes.
, -Carbonyldiimidazole (1.9 g, 11.0 mmol) is added and the mixture is stirred at room
temperature for 2 hours. The mixture is then heated to 55 °C and stirred an additional 18
hours. Water (50 mL) is added and the mixture is extracted with EtOAc (50 mL). The
aqueous layer is extracted with additional EtOAc (25 mL) and concentrated to dryness.
The crude residue is purified by silica gel flash column chromatography eluting with 50%
DCM in EtOAc, followed by 100% EtOAc. The appropriate fractions are concentrated
and dried under vacuum to give the title compound as a racemic cis mixture (1.33 g,
26%). ES/MS (m/z): 509 (M+H).
Preparation 18
N-((4aR,7aS)-6-Allyl-7a-(5-amino-2-fluoro-phenyl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d][l,3]thiazin-2-yl)benzamide, hydrochloride
NaOH (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-trifluoroacetyl)amino]phenyl]-4,4a,5,7-tetrahydropyrrolo[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 HCl (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). 1 N
HCl 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 19
N-((4aR,7aS)-7a-(5-Acetamido-2-fluoro-phenyl)-6-allyl-4,4a,5,7-tetrahydropyrrolo[3,4-
d][l,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-fluoro-phenyl)-4,4a,5,7-tetrahydropyrrolo[3,4-d][l,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 20
N-((4aR,7aS)-7a-(5-Acetamido-2-fluoro-phenyl)-4,4a,5,7-tetrahydropyrrolo[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 J-tetrahydropyrrolo[3,4-d][13]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 x
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%). H 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/MS (m/z): 413.1 (M+H).
Preparation 2 1
N-((4aR,7aS)-7a-(5-Acetamido-2-fluoro-phenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydro 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,5,7-tetrahydropyrrolo[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+H)+, H NMR (400 MHz, d6-DMSO) ppm 1.22 (t, = 7.28 Hz, 2 H) 1.92 - 2.07 ( ,
6 H) 2.89 - 3.20 (m, 2 H) 3.36 - 3.44 (m, 1 H) 3.67 (t, 7=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 22
(4aR,7aS)-7a-(5-Amino-2-fluoro-phenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydro rrolo[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-fluoro-phenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[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.
Alternate Preparation 22
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][l,3]thiazin-2-
amine (45.8 g, 125,3 mmol) in TFA (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 87 L (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 87 L (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 °C, and
neutralized with ammonium hydroxide 15% w/w. The aqueous layer is extracted with
EtOAc (3 x 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/z): 363 (M+H).
Preparation 23
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 dropwise 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/z): 280 (M+H).
Preparation 24
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 z): 234 (M+H).
Preparation 25
Benzyl N-allyl-N-[2-hydroxyiminoethyl]carbamate
A solution of benzyl N-allyl-N-(2-oxoethyl)carbamate (25 g, 107 mmol) in ACN
(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 ACN 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 26
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 dropwise 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/z): 247 (M+H).
Preparation 27
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
dropwise 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 z): ( Br/ 1Br) 421/423 (M+H).
Preparation 28
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 dropwise 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/z) : ( Br/ 1Br) 584/5 86 (M+H).
Preparation 29
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 HOAc (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/z): ( Br/ 1Br) 586/588 (M+H).
Preparation 30
Benzyl 2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d][1,3]thiazine- 6-carbox late
l,l'-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/z): ( Br/ 1Br). 568/570 (M+H)
Preparation 31
N-[7a-(5-Bromo-2-fluoro-phenyl)-4a,5,6,7-tetrahydro-4H-pyrrolo[3,4-d][l,3]thiazin-2-
yl]benzamide
Iodotrimethylsilane (2.21 mL, 15.46 mmol) is added dropwise 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 ACN (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/z): ( Br/ 1Br) 434/436 (M+H).
Preparation 32
feri-Butyl 2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d][l,3]thiazine-6-carboxylate
Di-?-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/z): ( Br/ 1Br) 534/536 (M+H).
Preparation 33
feri-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 ferf-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) and trans-N,N'-
dimethyl- 1,2-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 hours 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/z):
471 (M+H).
Preparation 34
N-[7a-(5-Amino-2-fluoro-phenyl)-4a,5,6,7-tetrahydro-4H-pyrrolo[3,4-d][l,3]thiazin-2-
yljbenzamide
TFA (10 mL) is added to a solution of ? -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 z): 371 (M+H).
Preparation 35
Racemic 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/z): 467 (M+H).
Preparation 36
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% ACN; 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 ACN: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 z): 467 (M+H).
Preparation 37
N-[3-[(4aR,7aS)-2-Benzamido-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, 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 1 N NaOH (2 x 50
mL), and with brine ( 1 x 50 mL). The silica gel purification is repeated as described
above to give the title compound (350 mg, 74%). ES/MS (m/z): 603 (M+H).
Preparation 38
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
(OD=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 DCM
(2.5 L) and water (5 L). The pH is adjusted to 1 with HC1 and the aqueous layer is
separated and neutralized with NaOH to adjust the pH to 10. The aqueous layer is
extracted with MTBE (3 x 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/z): 249 (M+H).
Preparation 39
l-Allyl-4-amino-4-(2-fluorophenyl)pyrrolidin-3-yl]methanol
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 (2 x 2 L). The aqueous layer is neutralized with
NaOH 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/z): 251 (M+H).
Preparation 40
[(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-p-toluoyl-L-tartaric acid (348.6 g, 884 mmol) in l-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 l-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/z): 251 (M+H).
Preparation 4 1
[(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 (21 g, 331 mmol) is dissolved in water (2.1 L) and
EtOAc (2.3 L). HC1 35% w/w is added to adjust the pH to 1. The aqueous layer is
separated and the pH adjusted to 10 with NaOH 50% w/w and extracted with EtOAc (2x).
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/z): 2 1 (M+H).
Preparation 42
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-
yljmethanol (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 x
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 z): 396 (M+H).
Preparation 43
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][l,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 1 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 x 800 mL). The pH of the aqueous layer is adjusted to 10 with
NaOH 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/z): 356 (M+H).
Preparation 44
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
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/z): 452 (M+H).
Preparation 45
(4aR,7aS)-7a-(2-Fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,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). HCl (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 x 200 mL).
The pH of the aqueous layer is adjusted with NaOH 50% w/w aqueous solution to pH=10
and extracted with EtOAc (3 x 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/z): 348 (M+H).
Preparation 46
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 hydrochloride.
N-[3-[(4aR,7aS)-2-Benzamido-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, 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 HC1 in diethyl ether (0.20 mL, 660 ) is added. The solvent is removed in vacuo
to give the title compound (7 mg, 23%). ES/MS (m/z): 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.
Preparation 47
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 , 10.2 mmol) is added to a solution of 5-
methoxypyrazine-2-carboxylic acid (1.6 g, 10.2 mmol) in ACN (53 mL) and DMF (848
). After 15 minutes, the freshly prepared solution is added to a solution of (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-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 NaOH 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) / DCM. 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/z): 499 (M+H).
Alternative Preparation of Crystalline Form 2 N-r3-[(4aR,7aS)-2-Amino-6-(5-
fluoropyrimidin-2-yl)-4,4a,5,7-tetrahvdropyrrolor3,4-dl[l,31thiazin-7a-yll-4-fluorophenyll-
5-methoxy-pyrazine-2-carboxamide (hydrated)
ACN (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/ACN solution. In a separate flask, the aqueous solution of
(4aR,7aS)-7a-(5-amino-241uoro-phenyl)-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 ACN
(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 dropwise 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+H), purity: 98.3%. H NMR (400 MHz, DMSO-i¾) ppm 2.99 -
3.07 (m, 2 H) 3.07 - 3.14 (m, 1 H) 3.58 - 3.67 (m, 1 H) 3.68 - 3.76 (m, 1 H) 3.76 - 3.84
(m, 1 H) 4.02 (s, 3 H) 4.07 (d, 7=10.92 Hz, 1 H) 6.08 (s, 2 H) 7.19 (dd, 7=11.98, 8.72 Hz,
1H) 7.78 - 7.89 (m, 2 H) 8.41 (s, 1 H) 8.44 (s, 2 H) 8.88 (s, 1H) 10.60 (s, 1 H).
General procedure for Preparation of Crystalline Form 2 N-r3-r(4aR,7aS)-2-Amino-6-(5-
fluoropyrimidin-2-yl)-4,4a,5,7-tetrahvdropyrrolor3,4-diri,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][l,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][l,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-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 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.
Angle Relative Intensity
Peak (2-Theta °) (% of most intense
+/- 0.2° peak)
1 11.8 100.0
2 18.6 71.4
3 19.3 45.5
4 26.7 41.9
5 20.6 27.3
6 9.0 19.0
7 24.8 18.5
8 22.4 15.5
9 31.9 14.3
10 10.6 11.2
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-methoxypyrazine-
2-carboxamide is the stable crystal form at room temperature and relative
humidity greater than about 15%.
Preparation 48
Crystalline Form 3 N-r3-r(4aR.7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-
4.4a.5.7-tetrahvdropyrrolor3.4-diri.31thiazin-7a-yll-4-fluoro-phenyll-5-methoxypyrazine-
2-carboxamide.
A thermogravimetric analysis pan is loaded 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 heated to about 170 °C and held at 170
°C for about 5 minutes. The mixture is cooled 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-tetrahvdropyrrolor3,4-diri.31thiazin-7a-yll-4-fluorophenyll-
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 (121 mg) is combined with ACN (5 mL) in a vial, and heated on a 90 °C stir
plate. 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, dried 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.
Example 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;
toluenesulfonic acid
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 (149.15 mg) is added to ethyl acetate (2 mL). The sample is
stirred at 1000 rpm at a temperature of 80°C. p-Toluenesulfonic acid (70 mg dissolved in
ethyl acetate ( 1 mL)) is added to the stirring solution, and it is stirred overnight at 80°C to
produce a slurry of a white solid which is isolated by vacuum filtration to provide the title
compound.
Alternative Preparation A 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; toluenesulfonic acid
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 (9.5 g, 19 mmol) and p-toluenesulfonic acid (3.80 g, 19.8 mmol) are added
to tetrahydrofuran (31 mL), water (7.9 mL), and 2-propanol (8.6 mL). The solution is
heated to 40 °C. To the warm solution is added 2-propanol (200.0 mL) over
approximately 3 hours. The mixture is seeded shortly after the start of the 2-propanol
addition with a portion of the title compound (500 mg, 0.75 mmol). After the solvent
addition is complete, the mixture is cooled to approximately 20 °C over 1-3 hours. The
mixture is heated from approximately 20 °C to approximately 55 °C over a target time of
2 hours. The temperature is held at 55 °C for 1 hour and then cooled to about 20 °C over
approximately 4 hours. The slurry is stirred for at least 10 hours at approximately 20 °C.
The slurry is filtered and the wet cake is washed with water (57 mL). The product is
dried in vacuo at 45 °C for at least 10 hours to give the title compound (10.4 g, 81%).
ES/MS (m/z): 500 (M+H).
Alternative Preparation B 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; toluenesulfonic acid
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 (20.7 g) is slurried at 170 rpm in 60:40 THF:H20 (85 mL) in a 500
mL 3-necked round bottomed flask equipped with a nitrogen bubbler, IKA® mechanical
motor/agitator attached to a glass shaft having a teflon banana blade, and a thermocouple
connected to a programmable J-KEM ® temperature controller. p-Toluenesulfonic acid
monohydrate (7.6 g, 1.03 eq) is dissolved in a mixture of 60:40 THF:H20 (20 mL) and
the solution added all at once to the stirring N-[3-[(4aR,7aS)-2-amino-6-(5-
fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]tliiazin-7a-yl]-4-fluorophenyl]-
5-methoxy-pyrazine-2-carboxamide slurry at 23 °C, leading almost immediately
to a clear reddish tan solution. The agitation rate is then increased to 200 rpm as over 15
minutes, water (22 mL) is added to the solution, which is then seeded 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-fluoro-phenyl]-5-methoxy-pyrazine-2-carboxamide
toluenesulfonic acid (750 mg, 3 wt % seed load) and is then stirred at 23 °C for a further
15 minutes. Over 6 hours, water (226 mL, total solvent of 353 mL; or 13.6 vol., final
solvent ratio of 17.5:82.5 THF:H20 ) is added to the slurry, which is then stirred overnight
(22 hours) at 23 °C. The slurry is filtered via vacuum, rinsed with 15:85 THF:H20 (2x20
mL), then left on vacuum for 20 minutes while cracks which form in the product wet cake
are manually pressed closed. The wet solids are dried at 40 °C under vacuum for about
72 hours to give the title compound as a white crystalline solid (24.07 g, 90.0 wt ).
The crystalline 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; toluenesulfonic acid is characterized by an XRD pattern using CuKa
radiation as having diffraction peaks (2-theta values) as described in Table 3 below, and
in particular having peaks at diffraction angle 2-theta of 5.0° in combination with one or
more of the peaks selected from the group consisting of 19.6°, 13.8°, and 18.5°; with a
tolerance for the diffraction angles of 0.2 degrees,
Table 3 : X-ray powder diffraction peaks of crystalline Example 1
Peak Angle (2-Theta°) +/- Relative Intensity ( of
0.2° most intense peak)
1 5.0 100.0
2 13.4 22.9
3 13.8 37.3
4 14.4 20.2
5 15.3 28.8
6 17.5 25.9
7 18.5 30.7
8 19.6 45.8
9 20.4 17.7
10 25.6 30.1
It has been found that below about 15% relative humidity, crystalline form 1 of
the free base of N-{3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4a,5,6,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-7a(4H)-yl]-4-fluoro-phenyl}-5-methoxypyrazine-2-
carboxamide is present, while above about 15% relative humidity, crystalline form 2 of
N-{3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4a,5,6,7-tetrahydropyrrolo[3,4-
d][l,3]thiazin-7a(4H)-yl]-4-fluoro-phenyl}-5-methoxypyrazine-2-carboxamide
predominates. Such physical instability is undesirable, particularly for a pharmaceutical
drug substance or drug product, both of which are subject to regulatory controls regarding
the solid state forms. In contrast to the crystalline forms of the free base, the tosylate salt
possesses improved physical stability over a wide range of relative humidities (5-95%).
As such, the tosylate salt of N-{3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-
4a,5,6,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-7a(4H)-yl]-4-fluoro-phenyl}-5-
methoxypyrazine-2-carboxamide essentially avoids the humidity dependent phase
transformations associated with the corresponding crystalline free base forms.
Furthermore, the tosylate salt of N-{3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-
4a,5,6,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-7a(4H)-yl]-4-fluoro-phenyl}-5-
methoxypyrazine-2-carboxamide was found to have a substantially lower amount of
solvent retained in the crystal of about 1.3% compared to about 7.7% solvent retained
for the HC1 salt of N-{3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4a,5,6,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-7a(4H)-yl]-4-fluoro-phenyl}-5-methoxypyrazine-2-
carboxamide. The increased likelihood of the free base to undergo phase transitions
during long term storage can lead to variability in properties, such as solubility or
dissolution rate, which may compromise the performance of the drug product. It also
poses the additional risk of collapse of the crystal structure(s) to an amorphous form or
accumulation of defects and disorder, which could lead to chemical instability and
reduced shelf-life of the drug product.
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 al.,
Science, 286, 735-741 (1999)). This fusion protein of BACEl(l-460) and human Fc,
named ¾i/BACEl:Fc, is constructed into the pJB02 vector. Human BACEl(l-460):Fc
( 2 BACEl :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 /2wBACEl :Fc .
2«BACEl: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 20x in KH2P0 4 buffer. Ten 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 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. al, 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 al, 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
ECso d IC 0 values. (See Sinha, et al., Nature, 402, 537-540 (2000)).
The compound of Preparation 46 is tested essentially as described above and
exhibited a BACE1 IC50 of 0.615 nM (+ 0.101, n=5) [Mean + SEM; SEM = standard error of
the mean]. This data demonstrates that the compound of Preparation 46 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/F12 (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, C1768). At day 5 in vitro, neurons are incubated at 37 °C for 24 hours 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 21F12 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 EC50 and IC50 values for the Abeta-lowering effect. The compound of Preparation 46
is tested essentially as described above and exhibited the following activity for Abeta
lowering effect:
Table 4
Mean + SEM; SEM = standard error of the mean
These data demonstrate that the compound of Preparation 46 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, PHARMASOLVE®, 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 al, Journal of euroscience, 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 Preparation
46, 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 vehicle-treated mice.
Given the activity of Preparation 46 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.
WE CLAIM:
1. A compound which is a tosylate salt 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.
2. The tosylate salt according to claim 1 which is crystalline.
3. The tosylate salt 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 5.0° in combination with one or more of the peaks selected from the
group consisting of 19.6°, 13.8°, and 18.5°; with a tolerance for the diffraction
angles of 0.2 degrees.
4. 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 3.
5. 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 to
3.
6. A compound according to any one of claims 1 to 3 for use in therapy.
7. A compound according to any one of claims 1 to 3 for use in the treatment of
Alzheimer's disease.
8. A compound according to any one of claims 1 to 3 for use in treating the
progression of mild cognitive impairment to Alzheimer's disease.
9. A pharmaceutical composition, comprising a compound according to any one
of claims 1 to 3 with one or more pharmaceutically acceptable carriers,
diluents, or excipients.