COMBINATION THERAPY
The present invention relates to a combination of a BACE inhibitor with an anti-
N3pGlu Abeta monoclonal antibody, and to methods of using the same to treat certain
neurological disorders, such as Alzheimer's disease.
The present invention is in the field of treatment of Alzheimer's disease and other
diseases and disorders involving amyloid b (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 beta-secretase (betasite
amyloid precursor protein-cleaving enzyme; BACE) has been shown to have a
significant effect on plaque-related and plaque-dependent pathologies in mouse models.
This suggests that even small reductions in Abeta peptide levels might result in a longterm
significant reduction in plaque burden and synaptic deficits, thus providing
significant therapeutic benefits, particularly in the treatment of Alzheimer's disease.
Moreover, antibodies that specifically target N3pGlu Abeta have been shown to
lower plaque level in vivo (US 2013/0142806). N3pGlu Abeta, also referred to as N3pE
or AbetaP3_42, is a truncated form of the Abeta peptide found only in plaques. Although
N3pGlu Abeta peptide is a minor component of the deposited Abeta in the brain, studies
have demonstrated that N3pGlu Abeta peptide has aggressive aggregation properties and
accumulates early in the deposition cascade.
A combination of a BACE inhibitor with an antibody that binds N3pGlu Abeta
peptide is desired to provide treatment for Abeta peptide-mediated disorders, such as
Alzheimer's disease, which may be more effective than either drug alone. For example,
treatment with such combination may allow for use of lower doses of either or both drugs
as compared to each drug used alone, potentially leading to lower side effects while
maintaining efficacy. It is believed that targeting the removal of deposited forms of
Abeta with an N3pG antibody and a BACE inhibitor will facilitate the phagocytic
removal of pre-existing plaque deposits while at the same time reduce or prevent further
deposition of Abeta by inhibiting the generation of Abeta.
US 2009/0209755 discloses fused aminodihydrothiazine derivatives which
possess BACE inhibitory activity and are further disclosed as useful therapeutic agents
for a neurodegenerative disease caused by Ab 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. U.S. Patent No. 8,278,334
discloses a method of treating a cognitive or neurodegenerative disease comprising
administering a substituted cyclic amine BACE-1 inhibitor with an anti-amyloid
antibody. Further, J. Neuroscience, 34(35), pages 11621-1 1630 (2014) discloses that
combined treatment with a BACE inhibitor and an anti-abeta antibody Gentenerumab
enhances amyloid reduction in APP L ondon mice.
Accordingly, the present invention provides a method of treating a cognitive or
neurodegenerative disease, comprising administering to a patient in need of such
treatment an effective amount of a BACE inhibitor in combination with an effective
amount of an anti-N3pGlu Abeta monoclonal antibody.
More specifically, the present invention provides a method of treating a cognitive
or neurodegenerative disease, comprising administering to a patient in need of such
treatment an effective amount of a com ound of Formula I :
Formula I
wherein R is H or F; and
A is:
or a pharmaceutically acceptable salt thereof; in combination with an effective amount of
an anti-N3pGlu Abeta monoclonal antibody.
The present invention also provides a method of treating a disease that is
characterized by the formation and deposition of Abeta, comprising administering to a
patient in need of such treatment an effective amount of a compound of Formula I, or a
pharmaceutically acceptable salt thereof, in combination with an effective amount of an
anti-N3pGlu Abeta monoclonal antibody.
The present invention further provides a method of treating Alzheimer's disease,
comprising administering to a patient in need of such treatment an effective amount of a
compound of Formula I, or a pharmaceutically acceptable salt thereof, in combination
with an effective amount of an anti-N3pGlu Abeta monoclonal antibody.
The present invention also provides a method of treating mild Alzheimer's
disease, comprising administering to a patient in need of such treatment an effective
amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in
combination with an effective amount of an anti-N3pGlu Abeta monoclonal antibody.
The present invention further provides a method of treating mild cognitive
impairment, comprising administering to a patient in need of such treatment an effective
amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, in
combination with an effective amount of an anti-N3pGlu Abeta monoclonal antibody.
The present invention further provides a method of treating prodromal
Alzheimer's disease, comprising administering to a patient in need of such treatment an
effective amount of a compound of Formula I, or a pharmaceutically acceptable salt
thereof, in combination with an effective amount of an anti-N3pGlu Abeta monoclonal
antibody.
In addition, the present invention provides a method for the prevention of the
progression of mild cognitive impairment to Alzheimer's disease, comprising
administering to a patient in need of such treatment an effective amount of a compound of
Formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective
amount of an anti-N3pGlu Abeta monoclonal antibody.
The present invention further provides a method of treating cerebral amyloid
angiopathy (CAA), comprising administering to a patient in need of such treatment an
effective amount of a compound of Formula I, or a pharmaceutically acceptable salt
thereof, in combination with an effective amount of an anti-N3pGlu Abeta monoclonal
antibody.
Furthermore, the present invention provides a compound of Formula I or a
pharmaceutically acceptable salt thereof, in combination with an effective amount of an
anti-N3pGlu Abeta monoclonal antibody, for use in therapy, in particular for the
treatment of Alzheimer's disease, mild Alzheimer's disease, prodromal 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 a
compound of Formula I, or a pharmaceutically acceptable salt thereof, with one or more
pharmaceutically acceptable carriers, diluents, or excipients, in combination with a
pharmaceutical composition of an anti-N3pGlu Abeta monoclonal antibody, with one or
more pharmaceutically acceptable carriers, diluents, or excipients.
In addition, the invention provides a kit, comprising a compound of Formula I, or
a pharmaceutically acceptable salt thereof, and an anti-N3pGlu Abeta monoclonal
antibody. The invention further provides a kit, comprising a pharmaceutical composition,
comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, with
one or more pharmaceutically acceptable carriers, diluents, or excipients, and a
pharmaceutical composition, comprising an anti-N3pGlu Abeta monoclonal antibody
with one or more pharmaceutically acceptable carriers, diluents, or excipients. As used
herein, a "kit" includes separate containers of each component, wherein one component is
a compound of Formula I, or a pharmaceutically acceptable salt thereof, and another
component is an anti-N3pGlu Abeta monoclonal antibody, in a single package. A "kit"
may also include separate containers of each component, wherein one component is a
compound of Formula I, or a pharmaceutically acceptable salt thereof, and another
component is an anti-N3pGlu Abeta monoclonal antibody, in separate packages with
instructions to administer each component as a combination.
The invention further provides the use of a combination of a compound of the
Formula I, or a pharmaceutically acceptable salt thereof, and an effective amount of an
anti-N3pGlu Abeta monoclonal antibody, for the manufacture of a medicament for the
treatment of Alzheimer's disease, mild Alzheimer's disease, prodromal Alzheimer's
disease or for the prevention of the progression of mild cognitive impairment to
Alzheimer's disease.
One of ordinary skill in the art will appreciate and recognize that "anti-N3pGlu
Abeta monoclonal antibody", and the specific antibodies, "B12L" and "R17L" are
identified and disclosed along with methods for making and using said antibodies by one
of ordinary skill in the art, in U.S. Patent No. 8,679,498 B2, entitled "Anti-N3pGlu
Amyloid Beta Peptide Antibodies and Uses Thereof, issued March 25, 2014 (USSN
13/810,895). See for example Table 1 of U.S. Patent No. 8,679,498 B2.
In addition, amino acid sequences for certain antibodies used in the present
invention are provided below in Table A:
Table A-Antibody SEQ ID NOs
A cognitive or neurodegenerative disease includes Alzheimer's disease, mild
Alzheimer's disease, mild cognitive impairment, prodromal Alzheimer's disease, cerebral
amyloid angiopathy (CAA), Down's syndrome, and the like.
As used herein, the terms "treating", "to treat", or "treatment", includes
restraining, slowing, stopping, reducing, or reversing the progression or severity of an
existing symptom, disorder, condition, or disease.
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 Formula I, or a pharmaceutically acceptable salt thereof, and to the amount
or dose of an anti-N3pGlu Abeta monoclonal antibody which, upon single or multiple
dose administration to the patient, provides the desired effect in the patient under
diagnosis or treatment. It is understood that the combination therapy of the present
invention is carried out by administering a compound of Formula I, or a pharmaceutically
acceptable salt thereof, together with the anti-N3pGlu Abeta monoclonal antibody in any
manner which provides effective levels of the compound of Formula I and the anti-
N3pGlu Abeta monoclonal antibody in the body.
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 Formula I and pharmaceutically acceptable salts thereof are
generally effective over a wide dosage range in the combination of the present invention.
For example, dosages per day normally fall within the range of about 0.1 mg/day to about
1000 mg/day, preferably about 0.1 mg/day to about 500 mg/day, and most preferably
about 0.1 mg/day to about 100 mg/day. In addition, the anti-N3pGlu Abeta monoclonal
antibody is generally effective over a wide dosage range in the combination of the present
invention. For example, dosages per week normally fall within the range of about 0.1 to
10 mg/kg/week, preferably about 0.3 to about 6 mg/kg/week, and most preferably about
0.3 mg/kg/week to about 3 mg/kg/week. In some instances dosage levels below the lower
limit of the aforesaid ranges 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 BACE inhibitors and the antibodies of the present invention are preferably
formulated as pharmaceutical compositions administered by any route which makes the
compound bioavailable. The route of administration may be varied in any way, limited
by the physical properties of the drugs and the convenience of the patient and the
caregiver. Preferably, anti-N3pGlu Abeta monoclonal antibody compositions are for
parenteral administration, such as intravenous or subcutaneous administration. In
addition, the BACE inhibitor, such as the compound of Formula I, or pharmaceutically
acceptable salt thereof, is for oral, parenteral, or transdermal administration, including
intravenous or subcutaneous administration. Such pharmaceutical compositions and
processes for preparing same are well known in the art. (See, e.g., Remington: The
Science and Practice of Pharmacy (D.B. Troy, Editor, 21st Edition, Lippincott, Williams
& Wilkins, 2006).
As used herein, the phrase "in combination with" refers to the administration of
the BACE inhibitor, such as a compound of Formula I, or a pharmaceutically acceptable
salt thereof, with an anti-N3pGlu Abeta monoclonal antibody, such as an anti-N3pGlu
Abeta monoclonal antibody simultaneously, or sequentially in any order, or any
combination thereof. The two molecules may be administered either as part of the same
pharmaceutical composition or in separate pharmaceutical compositions. The BACE
inhibitor can be administered prior to, at the same time as, or subsequent to administration
of the anti-N3pGlu Abeta monoclonal antibody, or in some combination thereof. Where
the anti-N3pGlu Abeta monoclonal antibody is administered at repeated intervals (e.g.
during a standard course of treatment), the BACE inhibitor can be administered prior to,
at the same time as, or subsequent to, each administration of the anti-N3pGlu Abeta
monoclonal antibody, or some combination thereof, or at different intervals in relation to
therapy with the anti-N3pGlu Abeta monoclonal antibody, or in a single or series of
dose(s) prior to, at any time during, or subsequent to the course of treatment with the anti-
N3pGlu Abeta monoclonal antibody.
The following paragraphs describe preferred groups, substituents, and
configurations of the present invention.
Preferred compounds are:
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; and
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]-3,5-difluoro-pyridine-2-
carboxamide; and the pharmaceutically acceptable salts thereof.
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, or a pharmaceutically acceptable salt thereof is especially preferred.
N-[3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrole [3,4-d] [1,3]thiazin-7a-yl] -4-fluoro-phenyl]-5-methoxy-pyrazine-2-
carboxamide is most preferred.
Furthermore, crystalline Form 2 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-
methoxy-pyrazine-2-carboxamide is a preferred compound; and crystalline Form 2 N-[3-
[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-4a,5,6,7-tetrahydropyrrolo[3,4-
d] [1,3]thiazin-7a(4H)-yl] -4-fluoro-phenyl]-5-methoxy-pyrazine-2-carboxamide,
characterized by a substantial peak in the X-ray diffraction spectrum, at diffraction angle
2-theta of 11.8°, with one or more 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.
Preferred anti-N3pGlu Abeta monoclonal antibodies are B12L and R17L which
are identified in U.S. Patent No. 8,679,498 B2 (See for example Table 1 therein), with
B12L being especially preferred.
One of ordinary skill in the art will appreciate that compounds of Formula I can
exist in tautomeric forms, as depicted in Scheme A. When any reference in this
application to one of the specific tautomers of the compound of Formula I is given, it is
understood to encompass both tautomeric forms and all mixtures thereof.
Scheme A
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 compound of Formula I 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.
One of ordinary skill in the art will appreciate that compounds of the invention are
comprised of a core that contains at least two chiral centers:
Scheme B
Although the present invention contemplates all individual enantiomers, as well as
mixtures of the enantiomers of said compounds, including racemates, the compounds
with the absolute configuration at the carbon atoms labeled 1 and 2 as illustrated in
Scheme B are preferred compounds of the invention.
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).
Compounds of Formula I, or pharmaceutically acceptable salts thereof, 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
procedures, to prepare compounds of Formula I, or salts thereof. The products of each
step can be recovered by conventional methods well known in the art, including
extraction, evaporation, precipitation, chromatography, filtration, trituration, and
crystallization. In addition, all substituents unless otherwise indicated, are as previously
defined. The reagents and starting materials are readily available to one of ordinary skill
in the art.
As used herein, "APP" refers to amyloid precursor protein; "BOC" refers to tertbutoxycarbonyl;
"BSA" refers to Bovine Serum Albumin; "CSF" refers to cerebrospinal
fluid; "DCC" refers to 1,3-dicyclohexylcarbodiimide; "DIC" refers to
diisopropylcarbodiimide; "DCM" refers to dichloromethane; "DIPEA" refers to
diisopropylethylamine "DMAP" refers to dimethylaminopyridine; "DMEM" refers to
Dulbecco's Modified Eagle's Medium; "DMSO" refers to dimethylsulfoxide; "EDCI"
refers to l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; "EDTA" refers
to ethylenediaminetetraacetic acid; "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;
"HATU" refers to (dimethylamino)-N,N-dimethyl(3H-[l,2,3]triazolo[4,5-ft]pyridin-3-
yloxy)methaniminium hexafluorophosphate; "HEK " refers to human embryonic kidney;
"hr refers to hour or hours; "HOAc" refers to acetic acid; "HOAt" refers to l-hydroxy-7-
azobenzotriazole; "HOBt" refers to 1-hydroxylbenzotriazole hydrate; "HBTU" refers to
refers to 2-(l H-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate;
"HRnR" refers to Horseradish Peroxidase; "IC50" refers to the concentration of an agent
that produces 50% of the maximal inhibitory response possible for that agent; "iPr" refers
to isopropyl; "min" refers to minute or minutes; "MTBE" refers to methyl t rt-butyl
ether; "PBS" refers to Phosphate Buffered Saline; "PDAPP" refers to platelet derived
amyloid precursor protein; "Prep" refers to preparation; "psi" refers to pounds per square
inch; "PyBOP" refers to benzotriazol-l-yloxytripyrrolidino-phosphonium
hexafluorophosphate; "PyBrop" refers to bromo-tris-pyrrolidino phosphoniumhexafluoro
phosphate; "RFU" refers to relative fluorescence unit "Rt" refers to retention time; "SCX"
refers to strong cation exchange chromatography; "SFC" refers to supercritical fluid
chromatography; "SEM" refers to standard error of the mean; "THF" refers to
tetrahydrofuran and "TMB" refers to 3,3',5,5'-teramethylbenzidine;.
The following preparations and examples further illustrate the invention.
In the schemes below, all substituents unless otherwise indicated, are as
previously defined. The reagents and starting materials are generally readily available to
one of ordinary skill in the art. Others may be made by standard techniques of organic
and heterocyclic chemistry using the procedures described in the Preparations and
Examples which follow including any novel procedures.
Scheme 1
Scheme 1 depicts the formation of oximes (product of Step 2) and (product of
Step 5). The oximes can each be used to form the bicyclic isoxazole (product of Steps 3
or 7). The substituted aromatic group can be inserted at different points of the synthesis
as shown in Scheme 1, Step 1 and Step 7. "PG" is a protecting group developed for the
amino group, such as carbamates and allyl. Such groups are well known and appreciated
in the art.
In a 2-step reaction, a ketone with a beta halogen can be alkylated (Step 1) with a
protected allyl amine using an inorganic base such as potassium carbonate and then
treated with hydroxylamine hydrochloride and an organic base such as pyridine in a polar
protic solvent such as ethanol to give the oxime (Step 2). The oxime product of Step 2
can then be converted to the bicyclic isoxazole in a 3+2 cyclization by several methods
such as heating the oxime of Step 2 in a non-polar solvent such as toluene or xylenes to
form the bicyclic isoxazole (Step 3). Alternatively, an oxime can be formed starting from
a dimethyl acetal which is treated with an acid such as formic acid to form the aldehyde
(product of Step 4). In step 5, the aldehyde of Step 4 can then be converted to the oxime
product of Step 5 with hydroxylamine hydrochloride and a base such as sodium acetate
trihydrate. The bicyclic isoxazole product of Step 6 can be formed from the oxime as
shown in Step 6 using an aqueous solution of sodium hypochlorite. In step 7, the
protected bicyclic isoxazole is then reacted with an aromatic organolithium reagent or
Grignard reagent to give protected bicyclic isoxazole product of Step 7.
Scheme 2
Scheme 2 illustrates different routes to the protected pyrrolo thiazine product of
Step 10 or Step 12. The protected bicyclic isoxazole can be treated with powdered Zn in
acetic acid or by Raney Nickel in a polar solvent such as ethanol under pressure
hydrogenation conditions to give an aminopyrrolidine methanol (product of Step 11).
The aminopyrrolidine methanol product of Step 11 is then reacted with benzoyl
isothiocyanate in a polar solvent such as THF followed by the addition of
l,lcarbonyldiimidazole (CDI) to give the fused protected pyrrolidine thiazine (product of
Step 12). Alternatively, the amine of the bicyclic isoxazole can be reacted with benzoyl
isothiocyanate to give the thiourea (product of Step 8), and then, in Step 9 the isoxazole
ring can be opened with powdered zinc in acetic acid to give the hydroxyl compound
product of Step 9. The hydroxyl compound can then be treated with CDI in a polar
aprotic solvent such as THF or l-chloro-N,N,2-trimethylpropenylamine in DCM to form
the fused protected pyrrolidine thiazine (product of Step 10). The fused pyrrolidine
thiazine can also be formed from a Mitsunobu reaction such as using triphenylphosphine
and diisopropyl azodicarboxylate (DIAD).
Scheme 3
Step 14 1. Deprotect
2. Heteroarylation
Scheme 3 depicts the conversion of the pyrrolo thiazine to the aniline (product of
Step 13) which can then be acylated followed by the deprotection and heteroarylation of
the pyrrolidine. Acylation of the aniline nitrogen and deprotection of the thiazine amine
leads to compounds of Formula I.
Azido-dehalogenation is performed on the appropriate pyrrolo thiazine in the
presence of an azide source, such as sodium azide. Such azido-dehalogenation reactions
are well known and appreciated in the art. Reduction of the resulting azide intermediate
to the aniline (product of Step 13) may be effected by hydrogenation conditions that are
well known and described in the art or by reducing agents well known in the art, such as
L1AIH4 , NaBH4, and PPh3.
A BOC protected pyrrolidine can be deprotected under acidic conditions well
known in the art (Step 1 of Step 14). The deprotected pyrrolidine can then be
heteroarylated in a nucleophilic aromatic substitution (SNAr) with a substituted aromatic
pyrimidine using an organic base such as dipea, triethylamine, or N,N,N,N' -
tetramethylguanidine to give the product of Step 2 in Step 14. The aniline product of
Step 14 can be coupled with heteroaromatic carboxylic acids under coupling conditions
(product of Step 1 of Step 15). One skilled in the art will recognize that there are a
number of methods and reagents for amide formation resulting from the reaction of
carboxylic acids and amines. For example, the reaction of an appropriate aniline with an
appropriate acid in the presence of a coupling reagent and an amine base such as DIPEA
or triethylamine, will give a compound of Formula I following deprotection of the
thiazine amine. Coupling reagents include carbodiimides such as DCC, DIC, EDCI, and
aromatic oximes such as HOBt and HOAt. Additionally, uronium or phosphonium salts
of non-nucleophilic anions such as HBTU, HATU, PyBOP, and PyBrOP can be used in
place of the more traditional coupling reagents. Additives such as DMAP may be used to
enhance the reaction. Alternatively, the protected aniline amine can be acylated using
substituted benzoyl chlorides in the presence of a base such as triethylamine or pyridine.
The protected thiazine amine can then be deprotected with an organic base such as
pyridine and methylhydroxylamine hydrochloride in a polar aprotic solvent such as
ethanol or an inorganic base such as lithium hydroxide in methanol to give compounds of
Formula I.
In an optional step, a pharmaceutically acceptable salt of a compound of Formula
I can be formed by reaction of an appropriate free base of Formula I with an appropriate
pharmaceutically acceptable acid in a suitable solvent under standard conditions.
Additionally, the formation of such salts can occur simultaneously upon deprotection of a
nitrogen protecting group. The formation of such salts is well known and appreciated in
the art. See, for example, Gould, P.L., "Salt selection for basic drugs," International
Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R.J., et al. "Salt Selection and
Optimization Procedures for Pharmaceutical New Chemical Entities," Organic Process
Research and Development, 4 : 427-435 (2000); and Berge, S.M., etal., "Pharmaceutical
Salts," Journal of Pharmaceutical Sciences, 66: 1-19, (1977). One of ordinary skill in the
art will appreciate that a compound of Formula I is readily converted to and may be
isolated as a pharmaceutically acceptable salt, such as a hydrochloride salt.
Preparations and Examples
The following preparations and examples further illustrate the invention.
Preparation 1
1-(3-Bromophenyl)-2-(diallylamino)ethanone
Potassium carbonate (38.8 g, 281 mmol) is added to 3-bromophenacyl bromide
(60 g 216 mmol) in acetonitrile (430 mL), and the mixture is cooled under nitrogen to 0
°C. Diallylamine (34.6 mL, 280.63 mmol) is added drop wise over 1 hour and the
reaction is allowed to warm to 22 °C overnight. The crude reaction mixture is
concentrated and the residue is partitioned in water (300 mL) and MTBE (300 mL). The
aqueous layer is discarded and the organic layer is washed with water (100 mL, 2 x) and
with brine (100 mL). The organic layer is dried over sodium sulfate, filtered, and the
solvent evaporated to constant weight to give the title compound (62 g, 98%). ES/MS
(m/e): 294 (M+l).
Preparation 2
Benzyl N-(2,2-dimethoxyethyl)carbamate
A solution of aminoacetaldehyde dimethyl acetal (25 mL, 229 mmol) in toluene
(120 mL) is treated at 0 °C with a 4.85 M sodium hydroxide solution (70.8 mL, 343.5
mmol). The mixture is stirred at 0 °C for 10 minutes and benzyl chloroformate (33.8 mL,
229 mmol) is added keeping the internal temperature below 20 °C during the addition.
The mixture is warmed to room temperature over 4 hours. The organic layer is separated,
washed with brine, dried over sodium sulfate, and concentrated to dryness to give the title
compound (54 g, 98%). ES/MS (m/e): 240 (M+H).
Preparation 3
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 4
Benzyl N-allyl-N-(2-oxoethyl)carbamate
o
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 5
1-(3-Bromophenyl)-2-(diallylamino)ethanone
A solution of l-(3-bromophenyl)-2-(diallylamino)ethanone (60 g, 204.7 mmol) in
ethanol (720 mL) and pyridine (24.8 mL, 307 mmol) is stirred 15 minutes at 22 °C.
Hydroxylamine hydrochloride (17 g, 246 mmol) is added in portions to the solution over
1 hour. The reaction is warmed to 50 °C for 2 hours and then heated to 70 °C for 16
hours. The solvent is evaporated and the residue partitioned in water (300 mL) and
MTBE (300 mL). The organic layer is separated and washed with water (100 mL, 2 x)
and brine (100 mL). The organic layer is dried over sodium sulfate, filtered, and
evaporated to dryness to give the title compound (75.5 g, 79%). ES/MS (m/e): 309
(M+l).
Preparation 6
2-(Diallylamino)- 1-(2-fluorophenyl)ethanone
Diallyl amine (1.56 L, 12.2 mol) is added to a solution of 2-bromo-l-(2-
fluorophenyl)ethanone (1291 g, 5.8 mol), in ethanol (12.9 L) keeping the internal
temperature below 30 °C. The reaction mixture is stirred for 4 hours at 22 °C.
Hydroxylamine hydrochloride (543 g, 7.58 mol) is added in portions to the solution and
then heated for 16 hours at 70 °C. The reaction is cooled to room temperature, the solvent
is evaporated, and the residue is partitioned in water (5.1 L) and MTBE (6.4 L). Sodium
carbonate is added to adjust the aqueous layer to pH= 5.5 and the aqueous layer is
extracted with additional MTBE ( 1.2 L). The organic layers are combined and washed
with water and brine. The organic layer is dried over sodium sulfate, filtered, and
evaporated to dryness to give the crude title compound (1.45 kg, 104%) which is used
without further purification. ES/MS (m/e): 249 (M+l).
Preparation 7
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 8
2-Bromo- 1-(5 -bromo-2-fluorophenyl)ethan- 1-one
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 9
5-Allyl-6a-(5-bromo-2-fluorophenyl)-l-(4-methoxybenzyl)hexahydro-lHpyrrolo[
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) d : 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 10
5-Allyl-6a-(3-bromophenyl)-3,3a,4,6-tetrahydro-lH-pyrrolo[3,4-c]isoxazole
The crude l-(3-bromophenyl)-2-(diallylamino)ethanone oxime (75.5 g, 195.34
mmol) is dissolved in toluene (600 mL) and refluxed for 12 hours. The solvent is
evaporated in vacuo and the residue dissolved in a mixture of aqueous 1N HCl ( 1 L) and
MTBE (300 mL). The mixture is stirred for 15 minutes and diatomaceous earth (10 g) is
added. The mixture is stirred for an additional 20 minutes and filtered through
diatomaceous earth. The filter cake is washed with additional aqueous 1N HCl (200 mL)
and MTBE (200 mL). The organic layer is separated and washed with 1N HCl (2 x 100
mL). The aqueous layers are combined and the pH adjusted to 9 with NaOH 50% w/w.
The aqueous mixture is extracted with MTBE (3 250 mL). The organic layers are
combined, dried over sodium sulfate, and filtered. The filtrate is evaporated and dried
under vacuum to give a red solid (60 g). The red solid is diluted with heptane (600 mL)
and the mixture heated to reflux for 20 minutes. Charcoal (2 g) is added and the mixture
is filtered through diatomaceous earth. The filtrates are concentrated under atmospheric
pressure to adjust the final volume to 300 mL. The solution is cooled to 22 °C and stirred
for 3 hours. A pale yellow solid is collected by filtration and dried under vacuum to a
constant weight to give the title compound (40 g, 60%). ES/MS (m/e): 309 (M+l).
Preparation 11
5-Allyl-6a-(2-fluorophenyl)-3,3a,4,6-tetraliydro-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 12
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 13
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 14
Benzyl 6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydro-lH-pyrrolo[3,4-c]isoxazole-5-
carboxylate
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 15
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-tetrahydro-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 16
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 17
(l-Allyl-4-amino-4-(5-bromo-2-fluorophenyl)pyrrolidin-3-yl)methanol
A saturated aqueous solution of sodium carbonate is added to a solution of 5-allyl-
6a-(5-bromo-2-fluorophenyl)hexahydro-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 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 18
[l-Allyl-4-amino-4-(3-bromophenyl)pyrrolidin-3-yl]methanol
A 22 °C solution of 5-allyl-6a-(3-bromophenyl)-3,3a,4,6-tetrahydro-lHpyrrolo[
3,4-c]isoxazole (40 g, 129.4 mmol) in acetic acid (400 mL) is treated with zinc
dust (42.3 g, 646.8 mmol) in one portion. The reaction is stirred vigorously at room
temperature for 1 hour. EtOAc (400 mL) is added and the mixture is filtered through
diatomaceous earth. The filtrate is evaporated and the residue dried under vacuum. The
residue is partitioned in water (300 mL) and MTBE (300 mL). The pH is adjusted to 8
with sodium hydroxide 50% w/w and the organic layer is separated, dried over sodium
sulfate, and filtered. The filtrate is evaporated and the residue is dried under vacuum to
give the title compound (41 g, 97%). ES/MS (m/e): 311 (M+l).
Preparation 19
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 20
[(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 m h;
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) d: 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 2 1
[(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 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 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 22
[(3R,4S)-l-Allyl-4-amino-4-(3-bromophenyl)pyrrolidin-3-yl]methanol; (2R,3R)-2,3-
bis[(4-methylbenzoyl)oxy]butanedioic acid
A solution of [l-allyl-4-amino-4-(3-bromophenyl)pyrrolidin-3-yl]methanol (77 g,
235 mmol) in isopropyl alcohol (914 mL) is heated to 70 °C. Di-/?-toluoyl-L-tartaric acid
(86.2 g, 223 mmol) is added and the mixture is cooled to 22 °C for 2 hours and stirred
overnight. The slurry is filtered to collect a pale yellow solid and washed with isopropyl
alcohol. The solid is dried under vacuum to give the title compound (63 g, 36%). ES/MS
(m e): 311 (M+l). The product is analyzed by reverse phase chiral chromatography:
Analysis of the first eluting isomer (Column: Chiralpak ID-3 4.6 x 50 mm; eluent: 70:30,
aqueous 20 mM ammonium bicarbonate: acetonitrile; flow: 1.5 mL/min at UV 215 nm)
confirms the enantiomerically enriched (96% ee) enantiomer with Rt = 1.26 minutes.
Preparation 23
((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 c 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 m h; 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 d: 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 24
[(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 25
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)metlianol (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 26
[(3R,4S)-l-Allyl-4-amino-4-(3-bromophenyl)pyrrolidin-3-yl]methanol
[(3R,4S)-l-Allyl-4-amino-4-(3-bromophenyl)pyrrolidin-3-yl]methanol; (2R,3R)-
2,3-bis[(4-methylbenzoyl)oxy]butanedioic acid (63 g 85.8 mmol) is combined with
aqueous 1N HC1 (800 mL) and EtOAc (400 mL) and the mixture is stirred for 15 minutes
at 22 °C. The layers are separated and the pH of the aqueous layer is adjusted to 10 with
sodium hydroxide 50% w/w. The aqueous mixture is extracted with MTBE (3 250
mL). The combined organic layers are dried over magnesium sulfate, filtered, and
evaporated to dryness to give the title compound (27 g, 99%). ES/MS (m e): 311 (M+1).
Preparation 27
N-[(4aR,7aS)-6-Allyl-7a-(3-bromophenyl)-4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-
2-yl]benzamide
A solution of [(3R,4S)-l-allyl-4-amino-4-(3-bromophenyl)pyrrolidin-3-
yljmethanol (27 g; 86.7 mmol) in THF (270 mL) is cooled to -5 °C under a nitrogen
atmosphere. Benzoyl isothiocyanate (12.3 mL, 9 1 mmol) is added drop wise keeping the
temperature below 0 °C. The reaction is allowed to warm to 22 °C for 1 hour. 1,1'-
Carbonyldiimidazole (28.1 g, 173.5 mmol) is added in a single portion and the reaction is
stirred for 1 hour at 22 °C and then heated to reflux for 16 hours. The solvent is removed
in vacuo and the residue is dried under vacuum. The crude material is partitioned in
MTBE (500 mL) and water (250 mL). The organic layer is separated, dried over
magnesium sulfate, filtered, and evaporated to dryness. The crude material is purified
over a silica gel gradient of 90/10 to 60/40 DCM / EtOAc to give the title compound (27
g, 68%). ES/MS (m e): 456 (M+l).
Preparation 28
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 m h; 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 29
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 30
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% ammionium 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 31
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 32
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
I,G-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 c
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 33
Benzyl 2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d] [1,3]thiazine-6-carboxylate
I ,G-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 34
N-((4aR,7aS)-7a-(5-Acetamido-2-fluorophenyl)-4,4a,5,6,7,7a-hexahydropyrrolo[3,4-
d][l,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) d: 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 35
N-[(4aR,7aS)-7a-(3-Bromophenyl)-4a,5,6,7-tetrahydro-4H-pyrrolo[3,4-d][l,3]thiazin-2-
yllbenzamide
A room temperature mixture of N-[(4aR,7aS)-6-allyl-7a-(3-bromophenyl)-
4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide ( 1 g, 2.19 mmol) andN,Ndimethylbarbituric
acid (0.868 g, 5.48 mmol) in chloroform (22 mL) is degassed by
bubbling nitrogen through the resulting slurry at room temperature for 5 minutes. The
mixture is treated with tetrakis(triphenylphosphine)palladium (0.261 g, 219 mihoΐ) and is
stirred for 1.5 hours under nitrogen. In a separate flask, a mixture of N-[(4aR,7aS)-6-
allyl-7a-(3-bromophenyl)-4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide
(22.2 g, 48.6 mmol) and N,N-dimethylbarbituric acid (19.28 g, 121.6 mmol) in
chloroform (486 mL) is degassed by bubbling nitrogen through the resulting slurry at
room temperature for 5 min. The mixture is treated with
tetrakis(triphenylphosphine)palladium (5.79 g, 4.86 mmol) and is stirred for 2 hours
under nitrogen. The two reactions are combined and the solvent is removed in vacuo to
give the crude product. The crude material is purified over silica gel with a 30 minute
0.5% to 10% methanol in DCM gradient to give the title compound (22.4 g, 100%).
ES/MS (m/e): (7 Br/ 1Br) 416/418 (M+H).
Preparation 36
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 c 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 37
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 38
t r t-Butyl (4aR,7aS)-2-benzamido-7a-(3-bromophenyl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d] [1,3]thiazine-6-carboxylate
A room temperature solution of N-[(4aR,7aS)-7a-(3-bromophenyl)
tetrahydro-4H-pyrrolo[3,4-d] [1,3]thiazin-2-yl]benzamide (22.4 g, 36.69 mmol) in DCM
(367 mL) is treated with di-t-butyldicarbonate (8.81 g, 40.36 mmol) followed by
triethylamine (7.67 mL, 55.04 mmol) and the reaction is stirred at room temperature for 1
hour under nitrogen. The solvent is removed in vacuo and the crude product is purified
over silica gel with a 25 minute 5% to 100% EtOAc in hexanes gradient to give the title
compound (20.22 g, 100%). ES/MS (m e): (7 Br/ 1Br) 516/518 (M+H).
Preparation 39
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-tetahydro-
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 40
t r t-Butyl (4aR,7aS)-7a-(3-aminophenyl)-2-benzamido-4,4a,5,7-tetrahydropyrrolo[3,4-
d] [1,3]thiazine-6-carboxylate
A solution of t r t-butyl (4aR,7aS)-2-benzamido-7a-(3-bromophenyl)
tetany dropyrrolo [3,4-d][l,3]thiazine-6-carboxy late (5 g, 9.7 mmol) and tans-N,N'-
dimethyl-l,2-cyclohexanediamine (220.3 mg, 1.5 mmol) in ethanol (100 mL) is treated
with sodium azide (1.30 g, 19.4 mmol). An aqueous solution of L-ascorbic acid sodium
salt (0.66 M, 3.2 mL, 2.1 mmol) and water (10 mL) is 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 and ice water is
added. The mixture is extracted with EtOAc (3x). The organic layers are combined and
dried over sodium sulfate, filtered, and the solvent is removed in vacuo to give crude
azide product. The crude azide product is combined with 10% palladium on carbon (2 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 2 hours. The reaction is vented and the mixture is filtered through diatomaceous earth
using DCM to rinse the filter cake. 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 (4 g, 91%). ES/MS (m/e): 453 (M+H).
Preparation 4 1
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- 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 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 42
t r t-Butyl (4aR,7aS)-2-benzamido-7a-[3-[(5-fluoropyridine-2-carbonyl)amino^
4,4a,5 ,7-tetrahydropyrrolo [3,4-d] [1,3]thiazine-6-carboxylate
A slurry of t r t-butyl (4aR,7aS)-7a-(3-aminophenyl)-2-benzamido-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazine-6-carboxylate (93 mg, 0.21 mmol), 5-
fluoropyridine-2-carboxylic acid (31.9 mg, 0.23 mmol), 1-hydroxybenzotriazole hydrate
(56.7 mg, 0.41 mmol) and EDCI (40 mg, 0.21 mmol) in DCM (4 mL) containing
dimethylformamide ( 1 ml) is treated with DIPEA (179.2 m , 1.03 mmol) and the
resulting mixture is stirred at room temperature overnight. The reaction mixture is diluted
with DCM (5 mL) and saturated aqueous sodium bicarbonate (15 mL). The organic layer
is separated and washed with saturated aqueous sodium chloride (10 mL), dried over
sodium sulfate, filtered, and the solvent removed in vacuo to give the crude title
compound (105 mg, 89%). ES/MS (m e): 576 (M+H).
Preparation 43
N-[3-[(4aR,7aS)-2-Benzamido-4a,5,6,7-tetrahydro-4H-pyrrolo[3,4-d][l,3]thiazin-7ayl]
phenyl]-5-fluoro-pyridine-2-carboxamide; 2,2,2-trifluoroacetic acid
t r t-Butyl (4aR,7aS)-2-benzamido-7a-[3-[(5-fluoropyridine-2-
carbonyl)amino]phenyl]-4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazine-6-carboxylate
(105mg, 0.18 mmol) is dissolved in DCM (2 mL) and treated with trifluoroacetic acid
(500 m , 6.6 mmol). The resulting yellow solution is stirred for 4 hours at room
temperature and the solvent removed in vacuo to give the crude title product (190 mg,
100%). ES/MS (m/e): 476 (M+H).
Preparation 44
N-[(4aR,7aS)-7a-(3-Aminophenyl)-4a,5,6,7-tetrahydro-4H-pyrrolo[3,4-d][l,3]thiazin-2-
yljbenzamide
Trifluoroacetic acid (25 mL) is added to a solution of t rt-butyl (4aR,7aS)-7a-(3-
aminophenyl)-2-benzamido-4,4a,5 ,7-tetrahydropyrrolo [3,4-d] [1,3]thiazine-6-carboxylate
(4 g, 8.84 mmol) in DCM (100 mL) and the mixture is stirred at room temperature under
nitrogen for 4 hours. The solvent is 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 (2.49 g, 80%). ES/MS (m e): 353 (M+H).
Preparation 45
N-[7a-(5-Arnino-2-fluoro-phenyl)-4a,5,6,7-te1rahydro-4H-pyrrolo[3,4-d][l,3]tliiazin-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 46
N-[(4aR,7aS)-7a-(3-Aminophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydro rrolo[3,4-d][1,3]thiazin-2-yl]benzamide
A solution of N-[(4aR,7aS)-7a-(3-aminophenyl)-4a,5,6,7-tetrahydro-4Hpyrrolo[
3,4-d][l,3]thiazin-2-yl]benzamide (2.49 g, 7.06 mmol), 5-fluoro-2-
chloropyrimidine (3.74 g, 28.26 mmol), and DIPEA (6.16 mL, 35.32 mmol) in 1,4-
dioxane (60 mL) is heated to reflux for 4 hours under nitrogen. The reaction is cooled,
diluted with water, and extracted with EtOAc (3 x). The combined organic extracts are
dried over sodium sulfate, filtered and the solvent is removed in vacuo to give the 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 (2.51 g, 79%). ES/MS (m/e): 449
(M+H).
Preparation 47
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
pyrrolo[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 48
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) d 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, 1H) 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., 1H) 10.46 - 10.59 (m, 1 H).
Preparation 49
(4aR,7aS)-7a-(5-Amino-2-fluorophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,6,7,7ahexahydro
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-acetainido-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 50
N-[3-[(4aR,7aS)-2-Benzamido-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl]phenyl]-5 -fluoro-pyridine-2-carboxamide
A solution of N-[3-[(4aR,7aS)-2-benzamido-4a,5,6,7-tetrahydro-4H-pyrrolo[3,4-
d] [1,3]thiazin-7a-yl]phenyl]-5-fluoro-pyridine-2-carboxamide; 2,2,2-trifluoroacetic acid
(150 mg, 254 mihoΐ), 5-fluoro-2-chloropyrimidine (68 mg, 5 1 mmol) and DIPEA (98 m ,
56 mihoΐ) is heated in DMSO (5 mL) overnight at 40 °C. Additional 5-fluoro-2-
chloropyrimidine (68 mg, 5 1 mihoΐ) and DIPEA (98 m , 56 mmol) is added and the
mixture is heated overnight at 50 °C. Additional 5-fluoro-2-chloropyrimidine (68 mg, 5 1
mihoΐ) and DIPEA (98 m , 56 mihoΐ ) is added and the mixture is heated overnight at 50 °C
for a third night. The reaction is cooled, diluted with saturated aqueous sodium carbonate
(50 mL) to give a slurry that is filtered and dried in a vacuum oven at 50 °C for 4 hours to
give the title compound (60 mg, 41%). ES/MS (m/e): 449 (M+H).
Alternate Preparation 50
N-[(4aR,7aS)-7a-(3-Aminophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide (282 mg, 628.73 mihoΐ) and 5-
fluoropyridine-2-carboxylic acid (106.46 mg, 754.47 mihoΐ) are combined in DCM (3
mL) and dimethylformamide (0.5 mL). HOBT ( 112.70 mg, 817.35 mihoΐ) and then EDCI
(159.07 mg, 817.35 mihoΐ) are added and the resulting mixture is stirred for 5 hours at
room temperature under nitrogen. The reaction mixture is diluted with water and the pH
is adjusted with 1N NaOH to -12. The mixture is extracted with EtOAc (3 *). The
organic extracts are combined, dried over sodium sulfate, filtered and the solvent
removed in vacuo to give the crude product. The crude product is purified over silica gel
with a 20 minute 5% to 100% EtOAc in hexanes gradient to give the title compound (327
mg, 91%). ES/MS (m e): 571 (M+H).
Preparation 51
N-[7a-(5-Arnino-2-fluoro-phenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydro rrolo[3,4-d][1,3]thiazin-2-yl]benzamide
A solution of N-[7a-(5-amino-2-fluoro-phenyl)-4a,5,6,7-tetraliydro
pyrrolo[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 52
N-[3-[(4aR,7aS)-2-Benzamido-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d] [1,3]thiazin-7a-yl]phenyl]-5-methoxy-pyrazine-2-carboxamide
N-[(4aR,7aS)-7a-(3-Aminophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide (400 mg, 891.81 mihoΐ ) and 5-
methoxypyrazine-2-carboxylic acid (165 mg, 1.07 mmol) are combined in DCM (4 mL)
and dimethylformamide (0.5 mL). HOBt (160 mg, 1.16 mmol) and then EDCI (226 mg,
1.16 mmol) are added and the resulting mixture is stirred for 5 hours at room temperature
under nitrogen. The reaction mixture is diluted with water and the pH is adjusted to -12
with 1N NaOH. The mixture is extracted with EtOAc (3 x). The combined organic
extracts are dried over sodium sulfate, filtered and the solvent removed in vacuo. The
crude product is purified over silica gel with a 20 minute 5% to 100% EtOAc in hexanes
gradient to give the title compound (482 mg, 92%). ES/MS (m/e): 585 (M+H).
Preparation 53
N-[3-[2-Benzamido-6-(5-fluoropyrimidin-2-yl)-4,4a,5 ,7-tetrahydropyrrolo [3,4-
d][l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-fluoro-pyridine-2-carboxamide
N-[7a-(5-A nino-2-fluoro-phenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide (302 mg, 647 mihoΐ) and 5-
fluoropyridine-2-carboxylic acid (110 mg, 777 mihoΐ) are combined in DCM (3 mL) and
dimethylformamide (0.5 mL). HOBT ( 116 mg, 842 mihoΐ) and then EDCI (164 mg, 842
mihoΐ) are added and the mixture is stirred overnight at room temperature under nitrogen.
The reaction mixture is diluted with water and the pH adjusted with 1N NaOH to -12
and then extracted with EtOAc (3 x). The organic layers are combined and filtered to
collect the insoluble material. The solids are washed with water and EtOAc and dried
under vacuum to give the title compound. The organic layer from the filtrate is dried over
sodium sulfate, filtered, and the solvent removed in vacuo. The residue is purified over
silica gel with a 20 minute 5% to 100% EtOAc in hexanes gradient to give additional title
compound with a combined yield (275 mg, 72%). ES/MS (m e): 590 (M+H).
Preparation 54
N-[(4aR,7aS)-7a-(5-A rnno-2-fluoro-phenyl)-6-(5-f uoropyrimidin-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 55
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 (0.361 g, 0.77 mmol, isomer 1) 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 56
N-[3-[(4aR,7aS)-2-Benzamido-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
te1rahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]phenyl]-5-cyano^yridine-2-carboxamide
N-[(4aR,7aS)-7a-(3-Aminophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide (0.30 g, 0.67 mmol) is dissolved in
DCM (10 mL) and 5-cyanopyridine-2-carboxylic acid (129 mg, 0.87 mmol), HOBt (185
mg, 1.34 mmol) and EDCI (169 mg, 0.87 mmol) are added. DIPEA (0.35 mL, 2 mmol) is
added and the reaction is stirred at room temperature overnight. The material is purified
directly with silica gel chromatography eluting with a 0-100% EtOAc / hexanes gradient
to give the title compound (360 mg, 88%). ES/MS (m e): 579 (M+H).
Preparation 57
N-[3-[(4aR,7aS)-2-Benzamido-6<5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl]phenyl]-3 ,5-difluoro-pyridine-2-carboxamide
N-[(4aR,7aS)-7a-(3-Aminophenyl)-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-2-yl]benzamide (0.30 g, 0.67 mmol) is dissolved in
DCM (10 mL) and 3,5-difluoropyridine-2-carboxylic acid (138 mg, 0.87 mmol), HOBT
(185 mg, 1.34 mmol) and EDCI (169 mg, 0.87 mmol) are added. Dipea (0.35 mL, 2
mmol) is added and the reaction is stirred at room temperature overnight. The reaction is
purified directly with silica gel chromatography eluting with a 0-100% EtOAc / hexanes
gradient to give the title compound (330 mg, 84%). ES/MS (m/e): 590 (M+H).
Preparation 58
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-cyano-pyridine-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 (0.180 g, 0.39 mmol, isomer 1) is
dissolved in a mixture of DCM (2 mL) and DMF (0.25 mL). 5-Cyanopyridine-2-
carboxylic acid ( 114 mg, 0.77 mmol), HOBt (106 mg, 0.77 mmol) and EDCI (150 mg,
0.77 mmol) are added and the reaction is stirred at room temperature overnight. The
mixture is diluted with water (10 mL), EtOAc (10 mL) and added to a solution of 1N
NaOH (100 mL). The mixture is extracted with EtOAc (2 x 100 mL) and the organic
layers are combined and washed with brine. The organic layer is dried over MgS0 4,
filtered, and concentrated. The residue is purified over silica gel chromatography using a
0-100% EtOAc / hexanes gradient to give the title compound (133 mg, 57%). ES/MS
(m e): 597 (M+H).
Preparation 59
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]-3,5-difluoro-pyridine-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 (0.180 g, 0.39 mmol, isomer 1) is
dissolved in a mixture of DCM (2 mL) and DMF (0.25 mL). 5-Cyanopyridine-2-
carboxylic acid ( 114 mg, 0.77 mmol), HOBt (106 mg, 0.77 mmol) and EDCI (150 mg,
0.77 mmol) are added and the reaction is stirred at room temperature overnight. The
mixture is diluted with water (10 mL) and EtOAc (10 mL) and then poured into a solution
of 1N NaOH (100 mL). The mixture is extracted with EtOAc (2 x 100 mL) the organic
extracts are combined and washed with brine. The organic layers are dried over MgS0 4,
filtered, and concentrated. The residue is purified via silica gel chromatography using a
0-100% EtOAc / hexanes gradient to give the title compound (190 mg, 80%). ES/MS
(m/e): 608 (M+H).
Preparation 60
(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 c 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 6 1
(4aR,7aS)-7a-(5-A rnno-2-fluoro-phenyl)-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 ° 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 A
N-[3-[2-A no-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-d][l ,3]thiazin-
7a-yl]-4-fluoro-phenyl]-5-fluoro-pyridine-2-carboxamide
A mixture of N-[3-[2-benzamido-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
te1rahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-fluoro-pyridine-2-
carboxamide (293 mg, 497 mihoΐ), O-methylhydroxylamine hydrochloride (430 mg, 4.97
mmol) and pyridine (402 m , 4.97 mmol) is heated in ethanol (13 mL) to 70 °C in a
capped flask for 2.5 hours. DMSO (3 mL) is added and the mixture is heated at 70 °C
overnight. Additional DMSO (10 mL) is added and heating continued at 70 °C for 4
hours. Additional O-methylhydroxylamine hydrochloride (208 mg, 2.48 mmol) and
pyridine (201 m , 2.48 mmol) is added and the mixture is heated to 60 °C for 3 hours and
the mixture is stirred for 3 days at room temperature. In a separate flask, a mixture of N-
[3-[2-benzamido-6-(5-fluoropyrimidin-2-yl)-4,4a,5 ,7-tetrahydropyrrolo [3,4-
d][l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-fluoro-pyridine-2-carboxamide (276 mg, 468
mihoΐ), O-methylhydroxylamine hydrochloride (405 mg, 4.68 mmol) and pyridine (478
m , 4.68 mmol) is heated in ethanol (15 mL) and DMSO (4 mL) at 70 °C in a capped
flask overnight. Additional DMSO (10 mL) is added and heating is continued at 70 °C
for 4 hours. Additional O-methylhydroxylamine hydrochloride (195 mg, 2.34 mmol) and
pyridine (189 x , 2.34 mmol) is added and heating continued at 70 °C for 3 hours
followed by stirring the mixture for 3 days at room temperature. The two reaction
mixtures are combined and most of the solvent removed in vacuo. The crude product is
purified on a SCX column using 3:1 DCM:methanol and then 2:1 DCM:7 N ammonia in
methanol. The crude product is further purified over silica gel with a 20 minute 0.5% to
10% gradient of 7 N ammonia methanol in DCM gradient to give the title compound (451
mg, 96%). ES/MS (m/e): 486 (M+H).
Example 1
N-{3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-4a,5,6,7-tetrahydropyrrolo[3,4-
d] [1,3]thiazin-7a(4H)-yl]phenyl} -5-fluoropyridine-2-carboxamide hydrochloride
A mixture of N-[3-[(4aR,7aS)-2-benzamido-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d] [1,3]thiazin-7a-yl]phenyl]-5-fluoro-pyridine-2-carboxamide (320
mg, 560 mihoΐ ), O-methylhydroxylamine hydrochloride (485 mg, 5.60 mmol) and
pyridine (453 m , 5.60 mmol) in ethanol (15 mL) is heated at 65 °C in a capped vial for
five hours. The reaction is cooled and the solvent removed in vacuo. The crude product
is purified over silica gel with a 30 minute 0.5% to 10% gradient of 7 N ammonia in
methanol DCM gradient to give N-[3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-
4,4a,5,7-tetrahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]phenyl]-5-fluoro-pyridine-2-
carboxamide (219 mg, 84%). This material is dissolved in DCM ( 1 mL) and methanol
(0.5 mL) and 1M hydrogen chloride in diethyl ether (0.47 mL, 470 mihoΐ ) is added. The
solvent is removed in vacuo to give the title compound (228 mg, 81%). ES/MS (m e):
468 (M+H).
Example 2
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]phenyl}-5-methoxypyrazine-2-carboxamide hydrochloride
A mixture of N-[3-[(4aR,7aS)-2-benzamido-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl]phenyl] -5-methoxy-pyrazine-2-carboxamide
(479 mg, 819 mhioΐ), O-methylhydroxylamine hydrochloride (709 mg, 8.19 mmol) and
pyridine (663 x , 8.19 mmol) in ethanol (20 mL) is heated at 50 °C in a capped flask
overnight. DMSO (4 mL) is added and the mixture is heated to 70 °C for 4 hours to
obtain a solution. The reaction is cooled and most of the solvent is removed in vacuo.
Water is added and the pH is adjusted to -12 with 1N sodium hydroxide. The mixture is
extracted with EtOAc (5 x). The combined organic extracts are dried over sodium
sulfate, filtered and the solvent removed in vacuo. The crude product is purified over
silica gel with a 30 minute 0.5% to 10% gradient of 7 N ammonia methanol in DCM
gradient. The mixture is purified again on a SCX column using 3:1 DCM:methanol and
then 2:1 DCM:7 N ammonia in methanol to remove residual DMSO. The mixture is
purified a final time over silica gel with a 20 minute 0.5% to 10% gradient of 7 N
ammonia methanol in DCM to give N-[3-[(4aR,7aS)-2-amino-6-(5-fluoropyrimidin-2-yl)-
4,4a,5 ,7-tetrahydropyrrolo [3,4-d] [1,3]thiazin-7a-yl]phenyl] -5-methoxy-pyrazine-2-
carboxamide. This material is dissolved in DCM ( 1 mL) and methanol (0.5 mL) and 1M
hydrogen chloride in diethyl ether (0.66 mL, 660 mihoΐ) is added. The solvent is removed
in vacuo to give the title compound (329 mg, 78%). ES/MS (m/e): 481 (M+H).
Example 3
N-[3-[(4aR,7aS)-2-Arnino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d] [1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-fluoro-pyridine-2-carboxamide hydrochloride
Racemic N-[3-[2-amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
tetrahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-fluoro-pyridine-2-
carboxamide (451 mg, 929 mihoΐ) is chirally purified by SFC (Column: Chiralcel OD-H
(5 um), 2.1 x 25 cm; eluent: 40 % methanol (0.2 % isopropylamine) in C0 2; flow 70
mL/min at UV 225 nm). Chiral analysis of the first eluting isomer: Column: Chiralcel
OD-H (5 mh ), 4.6 150 mm; eluent: 40% methanol (0.2% isopropylamine) in C0 2; flow
5 mL/min at UV 225 nm confirms the enantiomerically enriched (>99% ee) enantiomer
with Rt = 1.01 minutes (175 mg, 360 mihoΐ ). This material (free base, isomer 1) is
dissolved in DCM ( 1 mL) and methanol (0.5 mL) and 1M hydrogen chloride in diethyl
ether (0.36 mL, 360 mihoΐ ) is added. The solvent is removed in vacuo to give the title
compound (183 mg, 38%). ES/MS (m e) : 486 (M+H).
Example 4
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 (0.350 g, 0.58 mmol, isomer 1) is dissolved in THF (2 mL) and then
methanol (4 mL) and ethanol (4 mL) are added. 0-Methylhydroxylamine hydrochloride
(495 mg, 5.81 mmol) and pyridine (470 x , 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 mihoΐ ) is added. The solvent is
removed in vacuo to give the title compound (71 mg, 23%). ES/MS (m/e): 498 (M+H).
Example 5
Crystalline Form 2 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-methoxy-pyrazine-2-
carboxamide (hydrated).
Acetonitrile (500 mL) is added to dimethylformamide (19.2 mL, 248.9 mmol).
Oxalyl Chloride (39.3 g, 309.63 mmol) followed by 5-methoxypyrazine-2-carboxylic acid
(46.0g, 298.4 mmol) is added to the dimethylformamide solution. In a separate flask, the
aqueous solution of (4aR,7aS)-7a-(5 -amino-2-fluorophenyl)-6-(5 -fluoropyrimidin-2-yl)-
4,4a,5,6,7,7a-hexahydropyrrolo[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 acid 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 ( 1lOg 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) d 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).
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 l = 1.54060 A) and a Vantec
detector, operating at 35 kV and 50 mA. The sample is scanned between 4 and 40° in 2Q,
with a step size of 0.009° in 2Qand 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 2Qwill
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 ° 2Q), typically the
more prominent peaks. The crystal form diffraction patterns, collected at ambient
temperature and relative humidity, were adjusted based on NIST 675 standard peaks at
8.853 and 26.774 degrees 2-theta.
A prepared sample of crystalline Form 2 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-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 11.8°, with one or more 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 2 : X-ray powder diffraction peaks of crystalline Form 2 of Example 5.
Example 6
N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d][1,3]thiazin-7a-yl]phenyl]-5-cyano-pyridine-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]phenyl]-5-cyano-pyridine-2-carboxamide (360
mg, 0.59 mmol) is dissolved in ethanol (10 mL) and DCM (2 mL). OMethylhydroxylamine
hydrochloride (504 mg, 5.91 mmol) and pyridine (478 m , 5.91
mmol) are added and the reaction is stirred at room temperature over the weekend (70
hours). The reaction is warmed to 60 °C and stirred for 24 hours. The reaction is
concentrated to give the crude product and purified via silica gel chromatography using a
0-10% 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 1M hydrogen chloride in
diethyl ether (0.54 mL, 540 mihoΐ) is added. The solvent is removed in vacuo to give the
title compound (240 mg, 75%). ES/MS (m/e): 475 (M+H).
Example 7
N-[3-[(4aR,7aS)-2-Amino-6-(5-fluoropyriniidin-2-yl)-4,4a,5,7-tetrahydropyrrolo[3,4-
d][l,3]thiazin-7a-yl]phenyl]-3,5-difluoro-pyridine-2-carboxamide hydrochloride
N-[3-[(4aR,7aS)-2-Benzamido-6-(5-fluoropyrimidin-2-yl)-4,4a,5,7-
te1rahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]phenyl]-3,5-difluoro-pyridine-2-carboxamide
(330 mg, 0.53 mmol) is dissolved in THF (10 mL) and diluted with ethanol (10 mL). OMethylhydroxylamine
hydrochloride (453 mg, 5.32 mmol) and pyridine (430 m , 5.91
mmol) are added and the reaction is stirred at room temperature over the weekend (70
hours). The reaction is warmed to 60 °C and stirred for 24 hrs. The mixture is
concentrated onto silica gel (-10 g) and purified via silica gel chromatography using a 0-
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 1M hydrogen chloride in
diethyl ether (0.49 mL, 490 mihoΐ) is added. The solvent is removed in vacuo to give the
title compound (159 mg, 54%). ES/MS (m/e): 486(M+H).
Example 8
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-cyano-pyridine-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 -cyano-pyridine-2-
carboxamide (133 mg, 0.22 mmol, isomer 1) is dissolved in THF ( 1 mL) and diluted with
methanol (3 mL) and ethanol (3 mL). O-Methylhydroxylamine hydrochloride (190 mg,
2.2 mmol) and pyridine (180 m , 2.2 mmol) are added. The reaction is warmed to 50 °C
and stirred overnight. The mixture is concentrated onto silica gel (-10 g) and purified via
silica gel chromatography eluting with a 0-10% gradient of 7 N ammonia methanol in
DCM. The material 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 mixture 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 1M hydrogen
chloride in diethyl ether (0.27 mL, 270 mihoΐ) is added. The solvent is removed in vacuo
to give the title compound ( 114 mg, 97%). ES/MS (m e): 493 (M+H).
Example 9
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]-3,5-difluoro-pyridine-2-carboxamide
hydrochloride
-[3-[(4aR,7aS)-2-benzamido-6-(5 -fluoropyrimidin-2-yl)-4,4a,5 ,7-
te1rahydropyrrolo[3,4-d][l,3]thiazin-7a-yl]-4-fluoro-plienyl]-3,5-difluoro-pyridine-2-
carboxamide (190 mg, 0.31 mmol, isomer 1) is dissolved in THF ( 1 mL) and diluted with
methanol (3 mL) and ethanol (3 mL). O-Methylhydroxylamine hydrochloride (267 mg,
3.1 mmol) and pyridine (253 m , 3.1 mmol) are added and the reaction is warmed to 50
°C and stirred overnight. The reaction is purified on an SCX column using 3:1
DCM:methanol and then 2 :1 DCM:7 N ammonia in methanol. The material is purified a
final time over silica gel with a 0% to 10% gradient of 7 N ammonia in methanol in DCM
to give the free base of the title compound. This material is dissolved in DCM (5 mL)
and 1M hydrogen chloride in diethyl ether (0.20 mL, 200 mihoΐ) is added. The solvent is
removed in vacuo to give the title compound (101 mg, 60%). ES/MS (m/e): 504 (M+H).
Example 10
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]-3,5-difluoro-pyridine-2-carboxamide
Oxalyl chloride (10.5 ml, 136,4 mmol) is added to a solution of 3,5-
difluoropicolinic acid (19.9 g, 125 mmol) in acetonitrile (617 mL) and
dimethylformamide (10.5 mL). After 30 minutes stirring, the solution is added to a
freshly prepared solution of (4aR,7aS)-7a-(5-amino-2-fluoro-phenyl)-6-(5-
fluoropyrimidin-2-yl)-4,4a,5,7-te1rahydropyrrolo[3,4-d][l,3]thiazin-2-amine (41.1 g, 113
mmol) in a mixture ethanol (823 mL) / water (823 mL) previously heated to 50 °C. The
reaction is maintained at 50 °C for 1 hour. The reaction mixture is cooled to 22 °C and
the solvent is evaporated. The aqueous solution is diluted with DCM ( 1 L), and the pH is
adjusted with 2 M sodium hydroxide to pH=l 1. The organic layer is separated, and the
aqueous layer is washed with additional DCM (2 x 400 mL). The organic extracts are
combined, dried over sodium sulfate, filtered and evaporated to dryness. The crude
material is purified by silica gel chromatography using a gradient of ammoniated
methanol 2 N / DCM from 0-10% DCM to give the title compound as an off white solid
(45 g, 78%). ES/MS (m/e): 504 (M+l).
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 {See 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 mg 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.
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 20 in KH2R0 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, 1mg/mL Bovine
Serum Albumin, and 15 mM 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 uL 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, etal., Nature, 402, 537-540 (2000)).
The following exemplified compounds are tested essentially as described above
and exhibited the following activity for BACE1:
Table 3
Mean + SEM; SEM = standard error of the mean
These data demonstrate that the compounds of Table 3 inhibit purified
recombinant BACE1 enzyme activity in vitro.
Whole cell assays for measuring the Inhibition of Beta-Secretase Activity
HEK293Swe Whole Cell Assay
The routine whole cell assay for the measurement of inhibition of beta-secretase
activity utilizes the human embryonic kidney cell line HEK293p (ATCC Accession No.
CRL-1573) stably expressing a human APP751 cDNA containing the naturally occurring
double mutation Lys651Met652 to Asn651Leu652, commonly called the Swedish
mutation (noted HEK293Swe) and shown to overproduce Abeta (Citron, et ah, Nature,
360, 672-674 (1992)). In vitro Abeta reduction assays have been described in the
literature {See Dovey, etal., Journal ofNeurochemistry, 76, 173-181 (2001); Seubert, et
ah, Nature, 361, 260 (1993); and Johnson-Wood, et a , Proc. Natl. Acad. Sci. USA, 94,
1550-1555 (1997)).
Cells (HEK293Swe at 3.5xl0 4 cells/well, containing 200 mΐ culture media,
DMEM containing 10% FBS) are incubated at 37 °C for 4 to 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 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 following exemplified compounds are tested essentially as
described above and exhibited the following activity for Abeta lowering effect:
Table 4
These data demonstrate that the compounds of Table 4 inhibit native Abeta
production in whole cells.
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 mM (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 EC50 and IC50 values for the Abeta-lowering effect. The following exemplified
compounds are tested essentially as described above and exhibited the following activity
for Abeta lowering effect:
Table 5
Mean + SEM; SEM = standard error of the mean
These data demonstrate that the compounds of Table 5 inhibit 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 a , 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. Three hours after
administration of 1, 3, or 10 mg/kg oral dose of the compound of Example 1 to young
female PDAPP mice, Abeta 1-x peptide levels are reduced approximately 34%, 48%, and
53% in brain hippocampus, and approximately 43%, 59% and 66% in brain cortex,
respectively, compared to vehicle-treated mice.
Three hours after administration of 1 or 3 mg/kg oral dose of the compound of
Example 3, Abeta 1-x peptide levels are reduced approximately 38% and 50% in brain
hippocampus, and approximately 34% and 53% in brain cortex, respectively compared to
vehicle-treated mice.
For Example 4, 3 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 Examples 1, 3, and 4 against BACE enzyme in vitro, these
Abeta lowering effects are consistent with BACE inhibition in vivo, and further
demonstrate CNS penetration of Examples 1, 3, and 4.
These studies show that compounds of Examples 1 to 9 inhibit BACE and are,
therefore, useful in reducing Abeta levels.
Combination Study
BACE Inhibitor Feeding Pilot Study
A pilot pharmacokinetic and pharmacodynamic study is performed in PDAPP
mice fed a chow diet containing a BACE inhibitor, such as a compound of Formula I or
pharmaceutically acceptable salt thereof in order to define doses that provide minimal to
marked plasma and brain Abeta reduction by BACE inhibition alone. Young PDAPP
mice are fed for 14 days a diet containing a chow diet containing the BACE inhibitor at
"quasi-bid" equivalent doses of 3 mg/kg, 10 mg/kg, 30 mg/kg, or 100 mg/kg. The BACE
inhibitor at ~ 0.05, 0.15, 0.5, or 1.5 mg per gram of certified rodent diet #8728CM
(Harlan labs) is mixed in a Sorvall mixer for 10 minutes and then mixed with Hobart
mixer for 15 minutes prior to pelleting. Thirty-two young female PDAPP mice are
randomized by parental line into 4 groups of 8 consisting of a vehicle-treatment group
and the three doses of BACE inhibitor. Mice are allowed ad libitum access to food for 14
days and subsequently sacrificed. Mice are anesthetized with C0 2 and blood collected by
cardiac puncture into EDTA-coated microcentrifuge tubes and stored on ice.
Subsequently, plasma is collected by centrifugation of blood samples for 4 minutes at
14,000 rpm at room temperature, transferred to untreated microcentrifuge tubes, then
frozen on dry ice and stored at -80 °C until analysis. Mice are sacrificed by decapitation,
brains are rapidly micro-dissected into halves, flash frozen on dry ice and stored at -80 °C
until analysis (one half for Abeta analysis and the other half for compound exposures
measurement). For analysis of parenchymal Abeta, brain samples are homogenized in 5.5
M guanidine- HC1 buffer (0.5 mL per half brain) with tissue tearer (model 985-370) at
speed 5 for about 1 minute. Homogenized brain samples are nutated overnight at room
temperature.
For Abeta ELISA analysis, extracts are collected and diluted at least 1:10 in casein
buffer (lx PBS with 0.25% casein, 0.05% Tween 20, 0.1% thimerosal, pH 7.4 with
protease inhibitor cocktail (Sigma P9340 at 0.01 mg/mL)) and centrifuged at 14000 rpm
for 10 minutes. For analysis of plasma Abeta, samples are diluted 1:2 in specimen buffer
(PBS; 0.05% Triton X-405; 0.04% thimerasol, 0.6% BSA), prior to analysis by ELISA.
Plasma human Abetai_x is determined by sandwich ELISA using m266.2 (anti-Abetai3_28)
and biotinylated 3D6 (anti-Abetal-5) as the capture and reporter antibodies, respectively.
Unknowns are assayed in duplicate and pg/mL determined by interpolating (Soft Max Pro
v. 5.0.1, Molecular Dynamics, using 4-parameter fit of the reference curve) from 8 point
standard curves and then adjusting for dilution. Parenchymal Abeta is determined by
sandwich ELISAs as described above and the values are normalized to protein levels
(determined in duplicate by the Bradford Coomassie Plus Protein method) and expressed
as pg/mg protein.
To determine the tissue and plasma levels of the BACE inhibitor, the following
method is employed: A 0.1 mg/mL stock solution of BACE inhibitor is serially diluted
with methanol/water (1:1, v/v), to prepare working solutions, which are then used to
fortify control plasma and brain homogenates to yield analyte concentrations of 1, 5, 10,
20, 50, 100, 500, 1000, 2000, 4000, and 5000 ng/mL. Prior to analysis, brain samples are
homogenized in 3-volumes of methanol/water (1:4, v/v) with an ultrasonic disrupter. An
aliquot of each study sample, appropriate calibration standard and control matrix samples
are transferred to a 96-well plate and then mixed with acetonitrile containing internal
standard. After mixing, the samples are centrifuged to pellet the precipitated proteins.
Aliquots of the resulting supernatants are then transferred to a clean 96-well plate and
diluted with methanol/water (1:1, v/v), and 10 microliter aliquots are analyzed by LCMS/
MS. Analyte concentrations are calculated using the response to concentration
relationship determined by multiple regression of the calibration curve samples.
In Vivo Combination Study
In order to evaluate combinational plaque lowering therapy of an anti-N3pGlu
Abeta monoclonal antibody such as anti-N3pGlu- Abeta monoclonal antibody VII (see
Table 1, mE8c-IgG2a; as described in U.S. Patent No. 8,679,498 B2; USSN 13/810,895)
and a BACE inhibitor, such as a compound of Formula I or a pharmaceutically acceptable
salt thereof, a large cohort of PDAPP mice are first aged to 16 to 18-months of age. The
aged PDAPP mice are randomized into five treatment arms based upon gender, parental
line, and age. There are 20 to 30 aged PDAPP mice per treatment arm. Group 1 is
sacrificed as a time zero at study initiation in order to determine the baseline level of
pathology prior to therapeutic treatment (necropsy described below). The four remaining
groups are then treated as follows: Group-2, control animals receiving placebo chow diet
and weekly injections of 12.5 mg/kg of control isotype IgG2a antibody; Group-3, animals
receiving weekly injections of 12.5 mg kg anti-N3pGlu-Abeta monoclonal antibody;
Group-4, animals receiving BACE inhibitor chow diet at doses previously defined in the
pilot feeding study, but typically ~3 to 30 mg/kg/day; Group-5, animals receiving BACE
inhibitor chow diet (~3 to 30 mg/kg/day) and weekly injections of 12.5 mg/kg of anti-
N3pGlu-Abetamonoclonal antibody. The anti-N3pGlu-Abeta monoclonal antibody is
diluted from sterile stock solutions consisting of the antibody in PBS buffer and is
administered to the animals by intraperitoneal injections. The BACE inhibitor is mixed
with loose chow diet (-0.15 to 1.5 mg compound per gram of feed depending upon
desired dose) and compressed into feed pellets. Animal weight is recorded at study
initiation and subsequently weekly for the first month of treatment, and then monthly for
the study duration. The food intake is also monitored over the course of the study at
regular intervals. The animals receive the study treatments for a total of 4-months. The
animals stay on their respective diets until necropsy, which occurs one week after the
final antibody injections. At time of necropsy, the animals are anesthetized and blood
obtained by cardiac puncture using EDTA pre-rinsed 1ml syringes. The blood samples
are collected on ice and the plasma isolated by standard centrifugation. Subsequently, the
animals are perfused with cold heparinized saline and the brain removed and dissected
into the left and right hemi-spheres. One brain hemi-sphere is flash frozen and saved for
histological analyses. The remaining brain hemi-sphere is dissected into tissue segments
consisting of hippocampus, cortex, cerebellum, and mid-brain and subsequently frozen on
dry ice. The plasma and tissue samples are stored at -80°C until time of analysis.
Pharmacokinetic Evaluation
Plasma pharmacokinetics is determined on the plasma samples obtained at time of
necropsy. Plasma antibody levels are determined in an antigen binding ELISA assay
wherein plates are coated with antigen (AbetaP 3_42) and subsequently incubated with
diluted plasma samples or a reference standard consisting of a serial dilution of the anti-
N3pGlu monoclonal antibody in assay buffer (PBS + control murine plasma). After
washing the plate, the bound murine antibody was detected with an anti-murine-HRP
conjugated antibody followed by color development with TMB. To determine the tissue
(mid-brain) and plasma levels of the BACE inhibitor, the following method is employed:
A 0.1 mg/mL stock solution of BACE inhibitor is serially diluted with methanol/water
(1:1, v/v), to prepare working solutions, which are then used to fortify control plasma and
brain homogenates to yield analyte concentrations of 1, 5, 10, 20, 50, 100, 500, 1000,
2000, 4000, and 5000 ng/mL. Prior to analysis, brain samples are homogenized in 3-
volumes of methanol/water (1:4, v/v) with an ultrasonic disrupter. An aliquot of each
study sample, appropriate calibration standard and control matrix samples are transferred
to a 96-well plate and then mixed with acetonitrile containing internal standard. After
mixing, the samples are centrifuged to pellet the precipitated proteins. Aliquots of the
resulting supernatants are then transferred to a clean 96-well plate and diluted with
methanol/water (1:1, v/v), and 10 microliter aliquots are analyzed by LC-MS/MS.
Analyte concentrations are calculated using the response to concentration relationship
determined by multiple regression of the calibration curve samples.
Pharmacodynamic Evaluation
The parenchymal Abeta concentrations are determined in guanidine solubilized
tissue homogenates by sandwich ELISA. Tissue extraction is performed with the bead
beater technology wherein frozen tissue is extracted in 1ml of 5.5 M guanidine/50 mM
Tris/ 0.5X protease inhibitor cocktail at pH 8.0 in 2 ml deep well dishes containing 1ml
of siliconized glass beads (sealed plates were shaken for two intervals of 3-minutes each).
The resulting tissue lysates are analyzed by sandwich ELISA for Abetai_4 0 and Abetai -42:
bead beater samples are diluted 1:10 in 2% BSA/PBS-T and filtered through sample filter
plates (Millipore). Samples, blanks, standards, quality control samples, are further diluted
in 0.55 M guanidine/5 mM Tris in 2% BSA/PBST prior to loading the sample plates.
Reference standard are diluted in sample diluent. Plates coated with the capture antibody
21F12 (anti-Abeta42) or 2G3 (anti-Abeta^) at 15 mg/ml are incubated with samples and
detection is accomplished with biotinylated 3D6 (anti-Abetai_x) diluted in 2% BSA/PBST,
followed by 1:20 K dilution NeutrAvidin-HRP (Pierce) in 2% BSA/PBS-T and color
development with TMB (Pierce). The Abeta levels are interpolated from standard curves
and the final tissue concentration is calculated as nanograms of Abeta per milligram of
tissue wet weight. The percent area of the hippocampus and cortex occupied by
deposited Abeta is determined histologically. Cryostat serial coronal sections (7 to IOmih
thick) are incubated with 10 mg/ml of biotinylated 3D6 (anti-Abetai_x) or negative control
murine IgG (biotinylated). Secondary HRP reagents specific for biotin are employed and
the deposited Abeta visualized with DAB-Plus (DAKO). Immunoreactive Abeta deposits
are quantified in defined areas of interest within the hippocampus or cortex by analyzing
captured images with Image Pro plus software (Media Cybernetics).
These studies may show that the combination therapy of an anti-N3pGlu-Abeta
monoclonal antibody and a BACE inhibitor, such as a compound of Formula I or
pharmaceutically acceptable salt thereof, may result in enhanced Abeta reductions
relative to the individual mono-therapies.
Sequence Listing