Discovery of S3-Truncated, C-6 Heteroaryl Substituted Aminothiazine β-Site APP Cleaving Enzyme-1 (BACE1) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b01012

Truncation of the S3 substituent of the biaryl aminothiazine 2, a potent BACE1 inhibitor, led to a low molecular weight aminothiazine 5 with moderate activity. Despite its moderate activity, compound 5 demonstrated significant brain Aβ reduction in rodents. The metabolic instability of 5 was overcome by the replacement of the 6-dimethylisoxazole, a metabolic soft spot, with a pyrimidine ring. Thus, truncation of the S3 substituent represents a viable approach to the discovery of orally bioavailable, brain-penetrant BACE1 inhibitors.

Full text of DOI:10.1021/acs.jmedchem.6b01012

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Article
Discovery of S3-truncated, C-6 heteroaryl substituted
aminothiazine #-site APP cleaving enzyme-1 (BACE1) inhibitors
Yong-Jin Wu, Jason Guernon, Jianliang Shi, Lawrence Marcin, Mendi Higgins, Ramkumar
Rajamani, Jodi K. Muckelbauer, Hal A. Lewis, ChiehYing Chang, Dan Camac, Jeremy H.
Toyn, Michael K. Ahlijanian, Charles F. Albright, John E. Macor, and Lorin A. Thompson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01012 • Publication Date (Web): 25 Aug 2016
Downloaded from http://pubs.acs.org on August 28, 2016
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Discovery of S3-truncated, C-6 heteroaryl substituted aminothi-
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azine β-site APP cleaving enzyme-1 (BACE1) inhibitors
YongꢀJin Wu,*,a Jason Guernon,a Jianliang Shi,a Lawrence Marcin,a Mendi Higgins,a Ramkumar Raꢀ
jamani,a Jodi Muckelbauer,b Hal Lewis,b ChiehYing Chang,b Dan Camac,b Jeremy H. Toyn,a Michael
K. Ahlijanian,a Charles F. Albright,a John E. Macor,b and Lorin A. Thompson a
aResearch and Development, BristolꢀMyers Squibb Company, 5 Research Parkway, Wallingford, CT
06492ꢀ7660, USA
bPO Box 4000, Princeton, NJ 08543ꢀ4000, USA
Supporting Information Placeholder
ABSTRACT: Truncation of the S3 substituent of the biaryl aminothiazine 2, a potent BACE1 inhibitor,
led to a low molecular weight aminothiazine 5 with moderate activity. Despite its moderate activity,
compound 5 demonstrated significant brain Aβ reduction in rodents. The metabolic instability of 5 was
overcome by the replacement of the 6ꢀdimethylisoxazole, a metabolic soft spot, with a pyrimidine ring.
Thus, truncation of the S3 substituent represents a viable approach to the discovery of orally bioavailaꢀ
ble, brainꢀpenetrant BACE1 inhibitors.
INTRODUCTION
There is much evidence to suggest that βꢀamyloid (Aβ) peptide, particularly the longer 42 amino
acid form Aβ42, plays a critical role in the progression of Alzheimer’s disease (AD).1ꢀ3 For example,
human genetic mutations such as the Swedish mutation result in increased βꢀsecretase processing of βꢀ
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amyloid precursor protein (APP), elevated Aβ levels, and ultimately aggressive early onset AD.4
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more recent study reported a variant of the APP gene (APPA673T) that confers protection against AD in
certain humans. A673T substitution in APP reduces production of Aβ peptides secreted from heteroloꢀ
gously transfected cells, supporting the hypothesis that decreases in Aβ can protect against AD, and
conversely that increases in Aβ may underlie the pathology of AD.5 Aβ is derived from APP by proteꢀ
olysis. Cleavage of APP by βꢀsite APP cleaving enzymeꢀ1 (BACE1) results in shedding of the APP ecꢀ
todomain, and the remaining membrane bound Cꢀterminal fragment, C99, is further processed by γꢀ
secretase to produce Aβ. Thus, inhibition of BACE1 to reduce Aβ production is a promising approach
to test the amyloid hypothesis.6ꢀ10
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In 2009, Eli Lilly reported that 1 (LY2811376, Figure 1), a biaryl aminothiazine BACE1 inhibiꢀ
tor, demonstrated sustained brain Aβ reduction in healthy volunteers upon oral administration.11,12
However, this compound exhibited only modest enzyme inhibitory activity (IC50 = 0.27 ꢀM). Recently,
we disclosed a related series of compounds with potency improved by 34ꢀfold via introduction of a Cꢀ6
dimethylisoxazole substituent onto the thiazine core (compound 2, IC50 = 8 nM).13 This more potent
series benefits from the two hydrogenꢀbond interactions between the isoxazole and threonine 72 and
glutamine 73 of the flap backbone as revealed by the coꢀcrystal structure of 2 bound to the BACE1 acꢀ
tive site. Despite its potency, compound 2 produced only modest brain Aβ reduction in rodents.13 The
lack of robust in vivo activity was ascribed to poor brain penetration and metabolic instability of 2.13
Both hurdles were overcome by the replacement of the metabolically labile pyrimidine ring in 2 with a
methoxypyrazineꢀ2ꢀcarboxamide side chain to afford carboxamide 3 (Figure 1), which exhibited potent
BACE inhibitory activity (IC50 = 0.7 nM), and elicited robust brain Aβ reduction in rodents. Herein, we
describe another approach to address the two major issues confronting thiazine 2.
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Figure 1. Previous work
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RESULTS AND DISCUSSION
With regard to the modest brain penetration of 2, a common approach to improve brain penetraꢀ
tion is to reduce molecular weight and polar surface area. Thus, we probed truncation of the biaryl amiꢀ
nothiazine 2 by removing the pyrimidine ring in the S3 pocket. This approach was also expected to imꢀ
prove the metabolic stability, as in vitro studies had identified oxidation of the pyrimidine ring in 2 as a
metabolic soft spot. We prepared 5 from known bromide 413 in a straightforward fashion (Scheme 1).
The truncated aminothiazine 5 exhibited modest BACE1 inhibitory activity (IC50 = 150 nM). The 20ꢀ
fold reduction in potency, which was remarkably consistent with that observed in the 6ꢀunsubstituted
aminothiazine series,12 can be rationalized by examining the coꢀcrystal structures of both 213 and 514
bound to the BACE1 active site (Figure 2). The bicyclic aminothiazine core engaged the catalytic dyad
of BACE1 while the fluorophenyl ring bound in the S1 pocket; the dimethylisoxazole formed two hyꢀ
drogenꢀbonds with the flap backbone,15 and a potential CꢀH hydrogenꢀbond also exists between the Cꢀ6
hydrogen of the phenyl (see 5, Scheme 1) and the carbonyl of Gly230 on the backbone. The pyrimidine
ring of biaryl aminothiazine 2 is projected into the opening of the S3 pocket where it makes both hydroꢀ
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phobic contacts and interacts with the carbonyl of Ser229 via a bridging water molecule; consequently,
its removal reduces activity.
5
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Scheme 1. Synthesis of compound 5
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NHBoc
S
NH2
S
N
F
H
6
N
F
a,b
Br
O
N
O
N
F
F
4
5
IC50 = 150 nM
Reagents and conditions: (a) nꢀBuLi, THF, ꢀ78 °C, then H20 at rt, 67%; (b) TFA, CH2Cl2, rt, 57%.
Figure 2. Overlay of coꢀcrystal structures of 2 (magenta) and 5 (orange) bound with the BACE1 active
site.
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Despite its moderate potency, compound 5 produced a substantial pharmacodynamic effect, reꢀ
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ducing brain Aβ by 62% at 3 hours postꢀdose when administered orally to young mice at 30 mg/kg. The
total brain and plasma levels of 5 at 3 h postꢀdosing were 3.6 ꢀM and 4.8 ꢀM (brain/plasma: 0.75), reꢀ
spectively. In a similar experiment, biaryl aminothiazine 2 showed brain and plasma levels of 0.58 ꢀM
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and 7.4 ꢀM (brain/plasma: 0.08), respectively. Thus, the smaller analog 5 had a 6ꢀfold improvement in
brain exposure compared to 2. Oral administration of compound 5 to rats also produced a doseꢀ
dependent reduction of Aβ42 and total Aβ (Αβ1ꢀx) in brain (Figure 3), and the levels of reduction were
much more robust than those observed with the biꢀaryl aminothiazine 2 (the dose response data of 2 not
shown).
Compound 5 (mg/kg, PO)
Figure 3. Doseꢀdependent inhibition of Aβ40, Aβ42 and Aβ1ꢀx in the rat brain by compound 5 measured
at 5 h after oral administration (n = 5 rats/dose). p < 0.05.
While compound 5 showed improved in vivo activity relative to 2, its metabolic stability reꢀ
mained problematic. Compound 5 was relatively stable in rat and mouse liver microsomes, but unstable
in human liver microsomes (78% and 72% and 24% of parent remained after a 10 minute incubation
with mouse, rat and human liver microsomes, respectively, at 0.5 ꢀM). Interestingly, the nonꢀ
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fluorinated and monofluorinated analogs 6 and 7 exhibited superior stability in human liver microsomes,
but their BACE1 inhibitory activity was diminished (Figure 4). Due to their reduced activity, both comꢀ
pounds afforded only marginal mouse brain Aβ reduction when dosed orally to mice at 30 mg/kg. The
moderately active thiophene analog 8 displayed the worst metabolic stability profile.
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NH2
S
NH2
S
N
F
N
6
O
N
O
N
F
6, IC50 = 0.48
Metstab (H/R/M) = 86/81/78%
M
5, IC50 = 0.15
Metstab (H/R/M) = 24/78/72%
M
NH2
NH2
N
S
N
S
S
O
N
O
N
F
Cl
8, IC50 = 0.30
Metstab (H/R/M) = 11/12/21%
M
7, IC50 = 0.60
Metstab (H/R/M) = 73/78/90%
M
Figure 4. Analogs and metabolic stability (Metstab) in microsomes shown as % parent remaining after a
10 minute incubation at 0.5 ꢀM; H = human, R = rat, M = mouse.
The activity enhancement by the 6ꢀdimethylisoxazole group in the truncated series was clearly
demonstrated by compound 6, which is approximately 33ꢀfold more potent than its Cꢀ6 unsubstituted
counterpart 9 (IC50 = 22 ꢀM from the literatute12). This magnitude of increase was even larger than that
observed in the biaryl series (34ꢀfold, Figure 5).13 Overall, the trend was consistent in both series, but
the degree of the enhancement depended on the substituent(s) of the phenyl group.
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Figure 5. Activity enhancement by Cꢀ6 substitution.
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A major metabolic pathway of compound 5 involved oxidation of the methyl group(s) attached
to the isoxazole ring, and therefore, we attempted to block the metabolic soft spot(s) (Table 1). Reꢀ
placement of the isoxazole methyl with ethyl (10a/b), phenyl (10c/e), substituted phenyl (10d/f), pyridyl
(10g), and even the electronꢀwithdrawing difluoromethyl (10h) exerted little influence on either potency
or on metabolic stability. These substituents were positioned at the solvent interface and did not make
any significant interactions with the BACE1 active site, thus resulting in comparable activity. Within
this series, only the monomethyl substituted isoxazole analog 10i exhibited improved metabolic stability
across species. However, its modest in vivo activity prevented its further advancement.
Table 1. IC50 values of aminothiazinesa
Cpd
R1
R2
IC50
H/R/M
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(%)b
(ꢀM)
0.25
0.42
0.16
Me
Et
Et
Et
Ph
21/59/41
35/67/59
38/13/12
10a
10b
10c
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Me
pꢀFꢀ
Me
Ph
0.32
0.23
0.41
24/35/23
39/20/6
10d
10e
10f
Ph
Me
Me
pꢀFꢀ
45/88/53
Ph
Me
CHF2
H
3ꢀPy
Me
0.18
0.42
0.30
17/5/6
10g
10h
10i
35/67/59
65/77/80
Me
aAll values are the mean of at least two separate assay determinations. bpercent compound that remained
after a 10 minute incubation with (H)uman, (R)at or (M)ouse liver microsomes at 0.5 ꢀM.
Finally, we tackled our metabolic stability issue with 5 by replacing the entire dimethylisoxazole
group with other heterocycles, an exercise that led to two analogs 11 and 12 with improved metabolic
stability (Figure 6) and potency within 2ꢀfold of isoxazole analog 5. To understand if these heterocycles
behaved in the same fashion as the dimethylisoxazole, we obtained a coꢀcrystal structure of 12 bound
with the BACE1 active site16, and Figure 7 shows the overlay of coꢀcrystal structures of both 5 (orange)
and 12 (purple). Both molecules overlapped well, but the position of the flap backbone was different:
the flap was more open with pyrimidine analog 12 (BACE1 in green) than with isoxazole analog 5
(BACE1 in cyan). As a result, the dimethylisoxazole was closer to the flap than the pyrimidine (disꢀ
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tance from heteroatom of the heterocycle to the nitrogen of Thr72 or Glu 73: 3.4 Å for 5 versus 4.8 Å for
12). While their potencies were close, the data suggested that the pyrimidine bound primarily in the hy
-
5
6
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9
drophobic pocket near the flap region, and the isoxazole made more contact with the flap as a result of
the hydrogen bonds.
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NH2
S
NH2
S
N
F
N
N
N
N
N
F
F
F
11
, IC50 = 0.35 ꢀM
H/R/M = 70/78/72%
12
, IC50 = 0.31 ꢀM
H/R/M = 90/95/68%
Figure 6.
Figure 7. Overlay of coꢀcrystal structures of 5 (orange) and 12 (purple) bound with the BACE1 active
site. BACE1 in 5 is shown in green and BACE1 in 12 is shown in cyan.
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Compounds 11 and 12 produced improved results in our mouse Aβ reduction screen versus
compound 5. Using a dose of 10 mg/kg delivered orally, 11 produced a 62% reduction, and 12 produced
a 75% brain Aβ reduction. Both analogs demonstrated good plasma and brain drug levels (11, 4.3 ꢀM,
8
9
B/P = 1.3; 12, 6.7 ꢀM, B/P = 1.8). Because of its superior brain Aβ reduction in mice and good unꢀ
bound fraction across species (37%, 48% and 42% unbound in human, rat and mouse plasma), comꢀ
pound 12 was advanced to rat doseꢀresponse studies. As shown in Figure 8, this compound elicited a
doseꢀdependent reduction of Aβ40, Aβ42 and total Aβ1ꢀx in brain. The exposure of 12 was also measꢀ
ured in rat plasma and brain samples (Table 2) to allow quantification of its PK/PD relationship. Analyꢀ
sis of the rat brain Aβ42 reduction versus the total plasma concentration curve (Figure 9) resulted in the
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estimation of a brain Aβ42 EC50 of 3.8 ꢀM total plasma concentration or 1.8 ꢀM free drug level in
plasma (using rat plasma protein binding data), which was approximately 6ꢀfold the cellular IC50 value
of the compound (BACE1 IC50 = 0.31 ꢀM).
Compound 12 (mg/kg, PO)
Figure 8. Doseꢀdependent reduction of Aβ40, Aβ42 and Aβ1ꢀx in the brain by compound 12 measured
at 5 h after oral administration (N = 5 rats/dose). p < 0.05.
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Table 2. Brain, plasma concentration and brain/plasma ratio of 12a
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9
Dose
Plasma
Conc.
Brain
Conc.
Brain/plas
ma
(mg/k
g)
ratio
ꢀM (SD)
0.82 (0.13)
1.8 (1.3)
ꢀM (SD)
2.3 (0.3)
2.8 (1.7)
12 (3)
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2.8 (0.3)
1.8 (0.6)
1.8 (0.6)
10
6.6 (1.9)
aCompound dosed PO to female rats in PEGꢀ400:EtOH:Tween 80 (84:15:1); plasma and brain collected
5h postꢀdose (N=5 rats/dose) for determination of exposure and brain Aβ levels. Data are mean values
(N = 5) with SD in parentheses.
plasma concentration of 12
Figure 9. Brain Aβ42 reduction and plasma concentration curve of compound 12.
Compound 12 was administered to rats as either an IV bolus (2 mg/kg) or PO (10 mg/kg) to proꢀ
vide rat IV/PO pharmacokinetic data. It exhibited low clearance (12 mL/min/kg, approximately 20% of
rat hepatic blood flow) and a high volume of distribution (8.2 L/kg). The 10 mg/kg oral dose provided a
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Cmax of 3.7 ± 0.6 ꢀM at the 5 hour time point. The oral bioavailability based on the IV and PO AUC
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values was calculated to be more than 100%, potentially indicating nonlinear pharmacokinetics.
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The cytochrome P450 enzyme inhibitory potential of 12 was determined using recombinant
CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 enzymes in order to assess the potential likeliꢀ
hood of drug/drug interactions. Low levels of inhibition were observed across the major human P450
isozymes (IC50’s > 20 ꢀM) with the highest level of inhibition seen against CYP2C19 (IC50 = 9 ꢀM).17
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CHEMISTRY
Scheme 2 describes the synthesis of aminothiazine 12. Stereoselective addition of the lithium
enolate of methyl ketone 14 to Nꢀtertꢀbutanesulfinyl imine 13 gave the Nꢀsulfinyl βꢀamino ketone 15,
which underwent stereoselective reduction with lithium triꢀtertꢀ butoxyaluminum hydride to give synꢀ
1,3ꢀNꢀsulfinylꢀβꢀamino alcohol 16.18 Removal of the sulfinyl group led to the synꢀ1,3ꢀamino alcohol
17. Treatment of 17 with benzoyl isothiocyanate provided the benzoyl thiourea derivative 18, which
was converted to 12 under acidic conditions in moderate yield. Analogs 6ꢀ8, 10aꢀ10i, and 11 were preꢀ
pared following the same sequence.
Scheme 2. Synthesis of compound 12
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O
S
S
O
O
N
F
O
+
HN
a
N
N
N
F
F
F
N
13
14
15
O
S
9
c
OH
b
OH
H2N
HN
10
11
12
13
14
15
16
N
N
F
F
N
F
F
S
N
16
17
O
Ph
NH2
S
NH
N
OH
d
HN
e
17
N
N
18
19
20
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25
F
F
N
F
F
N
12
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Reagents and conditions: (a) LDA, THF, ꢀ78 to ꢀ30 °C, 44%; (b) LiAlH(OꢀtBu)3, ether, ꢀ78 to ꢀ30 °C,
93%; (c) 4N HCl in 1,4ꢀdioxane, MeOH, 100%; (d) benzoyl isothiocyanate, Hunig’s base, CH2Cl2, rt,
54%; (e) 1N H2SO4, 1,4ꢀdioxane, 100 °C, 6 h, 42%.
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CONCLUSIONS
we have developed a second approach to overcome the two major issues confronting biaryl amiꢀ
nothiazine 5, which were poor brain penetration and metabolic instability. Truncation of the S3 substitꢀ
uent improved brain penetration, but a metabolic stability issue still remained. This issue was resolved
through replacement of the 6ꢀdimethylisoxazole with a pyrimidine ring. This work suggested that trunꢀ
cation of the S3 substituent was a viable approach to the discovery of orally bioavailable, brainꢀ
penetrant BACE1 inhibitors.
EXPERIMENTAL SECTION
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General. Proton magnetic resonance (1 H NMR) spectra were recorded on either a Bruker
Avance 400 or a JEOL Eclipse 500 spectrometer and are reported in ppm relative to the reference solꢀ
vent of the sample in which they were run. HPLC and LC−MS analyses were conducted using a Shiꢀ
madzu SCLꢀ10A liquid chromatograph and a SPD UV−vis detector at 220 nm with the MS detection
performed with either a Micromass Platform LC spectrometer or a Waters Micromass ZQ spectrometer.
All flash column chromatography was performed on EM Science silica gel 60 (particle size of 40−60
μm). All reagents were purchased from commercial sources and used without further purification unless
otherwise noted. All reactions were performed under an inert atmosphere. HPLC analyses were perꢀ
formed using the following conditions. All final compounds had an HPLC purity of ≥95% unless specifꢀ
ically mentioned.
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HPLC Methods. Analytical HPLC analyses were carried out following methods A and B, and
preparatory reverseꢀphase scale purifications were performed using methods C and D.
Method A. A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (solvent A) and
95% acetonitrile, 5% water, and 0.05% TFA (solvent B) (t = 0 min, 10% B; t = 15 min, 100% B) was
employed on a SunFire C18 3.5 μm 3.5 mm × 150 mm column. Flow rate was 0.5 mL/min, and UV deꢀ
tection was set to 220 nm. The LC column was maintained at ambient temperature.
Method B. A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (solvent A) and
95% acetonitrile, 5% water, and 0.05% TFA (solvent B) (t = 0 min, 10% B; t = 15 min, 100% B (20
min)) was employed on a XBridge Ph 3.5 μm 3.0 mm × 150 mm column. Flow rate was 0.5 mL/min,
and UV detection was set to 220 nm. The LC column was maintained at ambient temperature.
Method C. Column: Waters XBridge C18, 19 x 200 mm, 5ꢀμm particles; Guard Column: Waꢀ
ters XBridge C18, 19 x 10 mm, 5ꢀμm particles; Mobile Phase A: water with 20ꢀmM ammonium acetate;
Mobile Phase B: 95:5 methanol:water with 20ꢀmM ammonium acetate; Gradient: 25ꢀ65% B over 40
minutes, then a 5ꢀminute hold at 100% B; Flow: 20 mL/min.
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Method D. Column: Waters Sunfire C18 OBD, 50 x 300 mm, 10ꢀμm particles; Guard Column:
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Waters Sunfire C18 OBD, 50 x 50 mm, 10ꢀμm particles; Mobile Phase A: 10:90 methanol:water with
0.1% trifluoroacetic acid; Mobile Phase B: 90:10 methanol:water with 0.1% trifluoroacetic acid; Gradiꢀ
ent: 0ꢀ100% B over 30 minutes, then a 5ꢀminute hold at 100% B; Flow: 150 mL/min.
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(4S,6S)-4-(2,4-Difluorophenyl)-6-(3,5-dimethylisoxazol-4-yl)-4-methyl-5,6-dihydro-4H-1,3-
thiazin-2-amine (5). Step A: To a solution of 4 (1 g, 1.936 mmol)13 in THF (19.36 mL) at ꢀ78 °C was
added dropwise nꢀBuLi (2.5M in hex) (3.10 mL, 7.75 mmol). The resulting mixture was stirred for 5
min. at ꢀ78 °C. The reaction mixture was then quenched by the addition of water (20 mL). The mixture
was then allowed to come to rt at which time it was diluted with ethyl acetate (50 mL), washed with waꢀ
ter (2 x 20 mL), brine (20 mL), dried over MgSO , filtered and concentrated in vacuo. Purification by
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silica gel flash chromatography eluting with 0ꢀ35% ethyl acetate/Hexanes) gave tertꢀbutyl ((4S,6R)ꢀ4ꢀ
(2,4ꢀdifluorophenyl)ꢀ6ꢀ(3,5ꢀdimethylisoxazolꢀ4ꢀyl)ꢀ4ꢀmethylꢀ5,6ꢀdihydroꢀ4Hꢀ1,3ꢀthiazinꢀ2ꢀ
yl)carbamate (581 mg, 67% yield) as a white foam. 1H NMR (400 MHz, CDCl3) δ 7.37 ꢀ 7.29 (m, 1H),
7.01 ꢀ 6.78 (m, 2H), 3.72 (dd, J = 13.1, 2.8 Hz, 1H), 2.87 (dd, J = 14.3, 2.8 Hz, 1H), 2.20 (s, 3H), 2.23
(1H, m), 1.76 (s, 3H), 1.53 (s, 9H). MS (M+H)+: 438.1. Step B: To a solution of tertꢀbutyl ((4S,6S)ꢀ4ꢀ
(2,4ꢀdifluorophenyl)ꢀ6ꢀ(3,5ꢀdimethylisoxazolꢀ4ꢀyl)ꢀ4ꢀmethylꢀ5,6ꢀdihydroꢀ4Hꢀ1,3ꢀthiazinꢀ2ꢀ
yl)carbamate (85 mg, 0.194 mmol) in DCM (3886 µl) was added TFA (388 µl, 5.04 mmol). The resultꢀ
ing mixture was stirred at rt for 3 h. The mixture was then concentrated in vacuo. Purification by flash
chromatography (Silica, 12g, 0 ꢀ 15% MeOH/CHCl3) gave a product which still contained an impurity.
Further purification by preparative HPLC (Method D) followed by basification of the appropriate comꢀ
bined fractions with NaHCO and extraction with ethyl acetate, drying (MgSO ) of the organic extract
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and concentration gave 5 (39.6 mg, 57% yield) as a clear, colorless oil. H NMR (500 MHz, CDCl3) δ
7.37 (td, J =9.1, 6.3 Hz, 1H), 7.04 ꢀ 6.83 (m, 2H), 3.85 (dd, J =12.9, 3.0 Hz, 1H), 2.90 (dd, J =14.3, 2.9
Hz, 1H), 2.30 (3H, s), 2.22 (3H, s), 2.14 (t, J =13.7 Hz, 1H), and 1.80 (3H, s). MS (M+H)+: 338.1.
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The absolute stereochemistry of this compound was confirmed by its coꢀcrystal structure with BACE1
active site.
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(4S,6S)-6-(3,5-Dimethylisoxazol-4-yl)-4-methyl-4-phenyl-5,6-dihydro-4H-1,3-thiazin-2-
amine (6). To a solution of Nꢀ(((2S,4R)ꢀ4ꢀ(3,5ꢀdimethylisoxazolꢀ4ꢀyl)ꢀ4ꢀhydroxyꢀ2ꢀphenylbutanꢀ2ꢀ
yl)carbamothioyl)benzamide (117 mg, 0.276 mmol) (prepared following the sequence described in Ref.
13) in dioxane (921 µl) was added HCl (5M aqueous) (4420 µl, 22.10 mmol). The resulting mixture
was brought to 90 °C and stirred for 1 h. The mixture was then allowed to come to rt and was quenched
by the addition of aqueous 1N NaOH until slightly basic. The mixture was then extracted with ethyl
acetate (3 x 3 mL). The combined extracts were washed with water (2 mL), brine (2 mL), dried over
MgSO4, filtered and concentrated in vacuo to give the crude oil. The crude material was purified via
preparative HPLC (Method C) to give 6 (40 mg, 48%). 1H NMR (500 MHz, DMSOꢀd6) δ 7.36 (d, J
=4.3 Hz, 4H), 7.30 ꢀ 7.17 (m, 1H), 3.56 (dd, J =12.7, 2.9 Hz, 1H), 2.46 (dd, J =13.6, 2.9 Hz, 1H), 2.20
(s, 3H), 2.08 (s, 3H), 1.92 (s, 3H), 1.82 (t, J =13.3 Hz, 1H). MS (M+H)+: 302.1.
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(4S,6S)-6-(3,5-Dimethylisoxazol-4-yl)-4-(4-fluorophenyl)-4-methyl-5,6-dihydro-4H-1,3-
thiazin-2-amine (7). B To a solution of Nꢀ(((2S,4R)ꢀ4ꢀ(3,5ꢀdimethylisoxazolꢀ4ꢀyl)ꢀ2ꢀ(4ꢀfluorophenyl)ꢀ
4ꢀhydroxybutanꢀ2ꢀyl)carbamothioyl)benzamide (28 mg, 0.063 mmol) (prepared following the sequence
described in Ref. 13) in dioxane (211 µl) was added HCl (5M aqueous) (1015 µl, 5.07 mmol). The reꢀ
sulting mixture was brought to 90 °C and stirred for 1 h. After coming to rt, the reaction mixture was
brought to pH 8 by the addition of 1 N aqueous NaOH. The mixture was then extracted with ethyl aceꢀ
tate (3 x 5 mL). The combined extracts were washed with water (3 mL), brine (3 mL), dried over
MgSO , filtered and concentrated in vacuo. The crude material was purified via preparative HPLC
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(Method C) to give 7 (4.6 mg, 0.014 mmol, 22.71 % yield) as a colorless oil. 1H NMR (500 MHz,
DMSOꢀd6) δ 7.48 ꢀ 7.40 (m, 2H), 7.32 (t, J =8.7 Hz, 2H), 3.93 ꢀ 3.79 (m, 1H), 2.96 ꢀ 2.86 (m, 1H), 2.50
ꢀ 2.43 (m, 1H), 2.28 (3H, s), 2.14 (3H, s) and 1.67 (3H, s). MS (M+H)+: 320.1.
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(4S,6S)-4-(3-Chlorothiophen-2-yl)-6-(3,5-dimethylisoxazol-4-yl)-4-methyl-5,6-dihydro-4H-
1,3-thiazin-2-amine (8).
To
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solution of Nꢀ(((2S,4R)ꢀ2ꢀ(3ꢀchlorothiophenꢀ2ꢀyl)ꢀ4ꢀ(3,5ꢀ
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dimethylisoxazolꢀ4ꢀyl)ꢀ4ꢀhydroxybutanꢀ2ꢀyl)carbamothioyl)benzamide (460 mg, 0.991 mmol) (preꢀ
pared following the sequence described in Ref. 13) in dioxane (10 mL) was added 6.0 M HCl (10 mL,
60.0 mmol). The mixture was stirred at 95 °C oil bath for 6 h. The mixture was concentrated, and satuꢀ
rated sodium bicarbonate was added. The aqueous layer was extracted with ethyl acetate, and the comꢀ
bined organic layes were dried over anhydrous sodium sulfate, and filtered. The filtrate was evaporated
in vacuo, and the residue was purified via silica gel chromatography eluting with 0ꢀ100% ethyl aceꢀ
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tate/hexanes to give 8 (120 mg, 35 % yield). H NMR (500 MHz, CDCl3) δ 7.18 (d, J =5.3 Hz, 1H),
6.92 (d, J = 5.5 Hz, 1H), 4.03 ꢀ 4.03 (m, 1H), 3.99 (dd, J =12.8, 3.1 Hz, 1H), 3.12 (dd, J = 13.9, 3.1 Hz,
1H), 2.32 (s, 3H), 2.24 (s, 3H), 1.80 (dd, J = 13.8, 13.0 Hz, 1H), 1.69 (s, 3H). MS (M+H)+: 342.05.
(4S,6S)-4-(2,4-Difluorophenyl)-6-(3-ethyl-5-methylisoxazol-4-yl)-4-methyl-5,6-dihydro-4H-
1,3-thiazin-2-amine (10a).
To a solution of Nꢀ(((2S,4R)ꢀ2ꢀ(2,4ꢀdifluorophenyl)ꢀ4ꢀ(3ꢀethylꢀ5ꢀ
methylisoxazolꢀ4ꢀyl)ꢀ4ꢀhydroxybutanꢀ2ꢀyl)carbamothioyl)benzamide (200 mg, 0.422 mmol) (prepared
following the sequence described in Ref. 13) in dioxane (1408 µl) was added HCl (5M aqueous) (6758
µl, 33.8 mmol). The resulting mixture was brought to 90 °C and stirred for 3h. The mixture was then
allowed to come to rt and was quenched by the addition of aqueous 1N NaOH until slightly basic. The
mixture was then extracted with ethyl acetate (3 x 3 mL). The combined extracts were washed with waꢀ
ter (2 mL), brine (2 mL), dried over MgSO , filtered and concentrated in vacuo to give the crude oil.
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The crude material was purified via preparative HPLC (Method C) to give 10a (27 mg, 18%). H NMR
(500 MHz, DMSOꢀd6) δ 7.37 ꢀ 7.21 (m, 2H), 7.13 (td, J = 8.5, 2.6 Hz, 1H), 3.56 (dd, J = 13.1, 3.1 Hz,
1H), 2.57 (dd, J =13.9, 2.9 Hz, 1H), 2.45 (qd, J = 7.6, 4.4 Hz, 2H), 1.90 ꢀ 1.82 (m, 1H), 1.56 (s, 3H),
1.03 (t, J = 7.5 Hz, 3H). MS (M+H)+: 352.0.
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(4S,6S)-6-(3,5-Diethylisoxazol-4-yl)-4-(2,4-difluorophenyl)-4-methyl-5,6-dihydro-4H-1,3-
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thiazin-2-amine (10b).
To
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difluorophenyl)ꢀ4 hydroxybutanꢀ2ꢀyl)carbamothioyl)benzamide (35 mg, 0.072 mmol) in dioxane (239
µl) was added HCl (5M aqueous) (1149 µl, 5.74 mmol). The resulting mixture was brought to 90 °C and
stirred for 3 h. The mixture was then allowed to come to rt and was quenched by the addition of aqueꢀ
ous 1N NaOH until slightly basic. The mixture was then extracted with ethyl acetate (3 x 3 mL). The
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combined extracts were washed with water (2 mL), brine (2 mL), dried over MgSO , filtered and conꢀ
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centrated in vacuo to give the crude oil. The crude material was purified via preparative HPLC (Method
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C) to give 10b (3.1 mg, 11% yield). H NMR (500 MHz, DMSOꢀd6) δ 7.36 ꢀ 7.20 (m, 2H), 7.14 (td, J
= 8.5, 2.6 Hz, 1H), 3.58 (d, J = 11.3 Hz, 1H), 2.66 (q, J = 7.6 Hz, 2H), 2.58 (d, J = 12.2 Hz, 1H), 2.48
(q, J = 7.6 Hz, 2H), 1.86 (m, 1H), 1.56 (s, 3H), 1.11 (3H,t, J = 7.6 Hz), 1.07 (3H, t, J = 7.6 Hz). MS
(M+H)+: 366.7.
(4S,6S)-4-(2,4-Difluorophenyl)-4-methyl-6-(5-methyl-3-phenylisoxazol-4-yl)-5,6-dihydro-
4H-1,3-thiazin-2-amine (10c). To a solution of Nꢀ(((2S,4R)ꢀ2ꢀ(2,4ꢀdifluorophenyl)ꢀ4ꢀhydroxyꢀ4ꢀ(5ꢀ
methylꢀ3ꢀphenylisoxazolꢀ4ꢀyl)butanꢀ2ꢀyl)carbamothioyl)benzamide (118 mg, 0.226 mmol) in dioxane
(1.2 ml) was added HCl (5M aqueous) (3.62 mL, 18.10 mmol). The resulting mixture was brought to
90 °C and stirred for 12 h. The mixture was then allowed to come to rt and was quenched by the addiꢀ
tion of aqueous 1N NaOH until slightly basic. The mixture was then extracted with ethyl acetate. The
combined extracts were washed with water (2 mL), brine (2 mL), dried over MgSO , filtered and conꢀ
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centrated in vacuo to give the crude oil which was purified by preparative HPLC (Method D) to afford
10c TFA salt (48 mg, 0.093 mmol, 40.9 % yield). 1H NMR (500 MHz, CD3OD) δ 7.52 ꢀ 7.46 (m, 1H),
7.43 ꢀ 7.37 (m, 2H), 7.28 ꢀ 7.17 (m, 3H), 7.11 ꢀ 6.98 (m, 2H), 4.01 (dd, J =12.9, 2.8 Hz, 1H), 3.03 (dd, J
= 14.7, 2.8 Hz, 1H), 2.65 (s, 3H), 2.62 ꢀ 2.54 (m, 1H), 1.84 (s, 3H). MS (M+H)+: 400.2.
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(4S,6S)-4-(2,4-Difluorophenyl)-6-(3-(4-fluorophenyl)-5-methylisoxazol-4-yl)-4-methyl-5,6-
dihydro-4H-1,3-thiazin-2-amine (10d). To a solution of Nꢀ(((2S,4R)ꢀ2ꢀ(2,4ꢀdifluorophenyl)ꢀ4ꢀ(3ꢀ(4ꢀ
fluorophenyl)ꢀ5ꢀmethylisoxazolꢀ4ꢀyl)ꢀ4ꢀhydroxybutanꢀ2ꢀyl)carbamothioyl)benzamide (118 mg, 0.219
mmol) (prepared following the sequence described in Ref. 13) in dioxane (2.5 mL) was added HCl (5M
aqueous) (3.50 mL, 17.50 mmol). The resulting mixture was brought to 90 °C and stirred for 16 h. The
mixture was then allowed to come to rt and was quenched by the addition of aqueous 1N NaOH until
nearly neutral (17 mL). The mixture was extracted with ethyl acetate. The combined extracts were
washed with water, brine, dried over magnesium sulfate, filtered and concentrated in vacuo. The crude
oil was purified using preparative HPLC (Method D) to afford 10d TFA salt (56 mg, 47% yield). 1H
NMR (500 MHz, CD3OD) δ 7.29 ꢀ 7.19 (m, 3H), 7.17 ꢀ 7.10 (m, 2H), 7.10 ꢀ 6.99 (m, 2H), 3.95 (dd, J =
12.8, 2.7 Hz, 1H), 3.00 (dd, J = 14.7, 2.8 Hz, 1H), 2.62 (s, 3H), 2.55 (dd, J =14.6, 13.0 Hz, 1H), 1.82 (s,
3H). MS (M+H)+: 418.1.
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(4S,6S)-4-(2,4-Difluorophenyl)-4-methyl-6-(3-methyl-5-phenylisoxazol-4-yl)-5,6-dihydro-
4H-1,3-thiazin-2-amine (10e). To a solution of Nꢀ(((2S,4R)ꢀ2ꢀ(2,4ꢀdifluorophenyl)ꢀ4ꢀhydroxyꢀ4ꢀ(3ꢀ
methylꢀ5ꢀphenylisoxazolꢀ4ꢀyl)butanꢀ2ꢀyl)carbamothioyl)benzamide (230 mg, 0.44 mmol) (prepared folꢀ
lowing the sequence described in Ref. 13) in dioxane (1.5 mL) was added HCl (5M aqueous) (7 mL,
35.3 mmol). The resulting mixture was brought to 90 °C and stirred for 16 h. The mixture was then
allowed to come to rt and was quenched by the addition of aqueous 1N NaOH until slightly basic. The
mixture was then extracted with ethyl acetate (3 x 100 mL). The combined extracts were washed with
water (25 mL), brine (25 mL), dried over MgSO , filtered and concentrated in vacuo to give 290 mg of
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the crude oil. The crude material was purified via preparative HPLC (Method C) to give 10e (44 mg,
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24%). H NMR (500 MHz, DMSOꢀd6) δ 7.32 ꢀ 7.24 (m, 1H), 7.18 (t, J = 7.6 Hz, 2H), 7.11 ꢀ 7.04 (m,
1H), 7.01 ꢀ 6.90 (m, 3H), 6.81 (t, J = 8.4 Hz, 1H), 3.62 (dd, J = 13.1, 2.4 Hz, 1H), 2.45 (d, J = 11.9 Hz,
1H), 2.18 (s, 3H), 1.75 (t, J = 13.4 Hz, 1H), 1.34 (s, 3H). MS (M+H)+: 400.3.
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(4S,6S)-4-(2,4-Difluorophenyl)-6-(5-(4-fluorophenyl)-3-methylisoxazol-4-yl)-4-methyl-5,6-
dihydro-4H-1,3-thiazin-2-amine (10f). To a solution of Nꢀ(((2S,4R)ꢀ2ꢀ(2,4ꢀdifluorophenyl)ꢀ4ꢀ(5ꢀ(4ꢀ
fluorophenyl)ꢀ3ꢀmethylisoxazolꢀ4ꢀyl)ꢀ4ꢀhydroxybutanꢀ2ꢀyl)carbamothioyl)benzamide (473 mg, 0.877
mmol) (prepared following the sequence described in Ref. 13) in dioxane (2.9 mL) was added HCl (5M
aqueous) (14 mL, 70.0 mmol). The resulting mixture was brought to 90 °C and stirred for 16h. The
mixture was then allowed to come to rt and was quenched by the addition of aqueous 1N NaOH until
slightly basic. The mixture was then extracted with ethyl acetate (3 x 100 mL). The combined extracts
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were washed with water (25 mL), brine (25 mL), dried over MgSO , filtered and concentrated in vacuo
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to give 290 mg of the crude oil. The crude material was purified via preparative HPLC (Method C) to
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give 10f (51.8 mg, 14% yield). H NMR (500 MHz, DMSOꢀd6) δ 7.42 ꢀ 7.27 (m, 5H), 7.25 ꢀ 7.17 (m,
1H), 7.11ꢀ6.94 (m, 1H), 3.77 (d, J =13.1 Hz, 1H), 2.67 (d, J =14.0 Hz, 1H), 2.40 (s, 3H), 1.95 (m, 1H),
1.56 (s., 3H). MS (M+H)+: 418.3.
(4S,6S)-4-(2,4-Difluorophenyl)-4-methyl-6-(5-methyl-3-(pyridin-3-yl)isoxazol-4-yl)-5,6-
dihydro-4H-1,3-thiazin-2-amine (10g). To a solution of Nꢀ(((2S,4R)ꢀ2ꢀ(2,4ꢀdifluorophenyl)ꢀ4ꢀ
hydroxyꢀ4ꢀ(5ꢀmethylꢀ3ꢀ(pyridinꢀ3ꢀyl)isoxazolꢀ4ꢀyl)butanꢀ2ꢀyl)carbamothioyl)benzamide (41 mg, 0.078
mmol) (prepared following the sequence described in Ref. 13) in dioxane (262 µl) was added HCl (5M
aqueous) (1255 µl, 6.28 mmol). The resulting mixture was brought to 90 °C and stirred for 4 h. The
mixture was then allowed to come to rt and was quenched by the addition of aqueous 1N NaOH until
slightly basic. The mixture was then extracted with ethyl acetate (3 x 100 mL). The combined extracts
were washed with water (25 mL), brine (25 mL), dried over MgSO , filtered and concentrated in vacuo.
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The crude material was purified via preparative HPLC (Method C) to give 10g (21 mg, 65%). H NMR
(500MHz, DMSOꢀd6) δ 8.67 (d, J = 3.4 Hz, 1H), 8.45 (s, 1H), 7.58 (d, J = 7.3 Hz, 1H), 7.48 ꢀ 7.39 (m,
1H), 7.21 ꢀ 7.08 (m, 2H), 7.00 (t, J = 8.7 Hz, 1H), 3.62 (1H, m), 2.63 (d, J = 13.4 Hz, 1H), 1.87 ꢀ 1.78
(m, 1H), 1.52 (s, 3H). MS (M+H)+: 401.0.
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(4S,6S)-6-(5-(Difluoromethyl)-3-methylisoxazol-4-yl)-4-(2,4-difluorophenyl)-4-methyl-5,6-
dihydro-4H-1,3-thiazin-2-amine (10h).
To a solution of Nꢀ(((2S,4R)ꢀ4ꢀ(5ꢀ(difluoromethyl)ꢀ3ꢀ
methylisoxazolꢀ4ꢀyl)ꢀ2ꢀ(2,4ꢀdifluorophenyl)ꢀ4ꢀhydroxybutanꢀ2ꢀyl)carbamothioyl)benzamide (100 mg,
0.20 mmol) (prepared following the sequence described in Ref. 13) in dioxane (1.5 mL) was added HCl
(5M aqueous) (3.2 mL, 16.2 mmol). The resulting mixture was brought to 90 °C and stirred for 16 h.
The mixture was concentrated under high vacuum on the rotory evaporator. The residue was purified
using preparative HPLC (Method D) to afford 10h TFA salt (3.7 mg, 7.21 µmol, 4 % yield) as a clear
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residue. H NMR (500 MHz, CD3OD) δ 7.39 ꢀ 7.30 (m, 1H), 7.20 ꢀ 6.97 (m, 3H), 4.40 (dd, J =13.1,
3.4 Hz, 1H), 3.08 (dd, J =14.6, 3.4 Hz, 1H), 2.59 ꢀ 2.49 (m, 1H), 2.41 (s, 3H), 1.84 (s, 3H). MS
(M+H)+: 374.1.
(4S,6S)-4-(2,4-Difluorophenyl)-4-methyl-6-(3-methylisoxazol-4-yl)-5,6-dihydro-4H-1,3-
thiazin-2-amine (10i.)
To a solution of Nꢀ(((2S,4R)ꢀ2ꢀ(2,4ꢀdifluorophenyl)ꢀ4ꢀhydroxyꢀ4ꢀ(3ꢀ
methylisoxazolꢀ4ꢀyl)butanꢀ2ꢀyl)carbamothioyl)benzamide (40 mg, 0.09 mmol) in dioxane (0.3 mL) was
added HCl (5M aqueous) (1.4 mL, 7.2 mmol). The resulting mixture was brought to 90 °C and stirred
for 16 h. The mixture was then allowed to come to rt and was quenched by the addition of aqueous 1N
NaOH until slightly basic. The mixture was then extracted with ethyl acetate (3 x 100 mL). The comꢀ
bined extracts were washed with water (25 mL), brine (25 mL), dried over Na2SO4, filtered and concenꢀ
trated in vacuo. The crude material was purified via preparative HPLC (Method C) to give 10i (4.6 mg,
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16 % yield). H NMR (500 MHz, DMSOꢀd6) δ 8.82 (s, 1H), 7.36 ꢀ 7.05 (m, 3H), 3.65 (d, J = 12.8 Hz,
2H), 2.74 (br. s., 2H), 2.13 (s, 3H), 1.75 (t, J = 13.4 Hz, 1H), 1.55 (s, 3H). MS (M+H)+: 324.3.
(4S,6S)-4-(2,4-Difluorophenyl)-4-methyl-6-(1-methyl-1H-pyrazol-5-yl)-5,6-dihydro-4H-1,3-
thiazin-2-amine (11). To a solution of Nꢀ(((2S,4R)ꢀ2ꢀ(2,4ꢀdifluorophenyl)ꢀ4ꢀhydroxyꢀ4ꢀ(1ꢀmethylꢀ1Hꢀ
pyrazolꢀ5ꢀyl)butanꢀ2ꢀyl)carbamothioyl)benzamide (134 mg, 0.30 mmol) (prepared following the seꢀ
quence described in Ref. 13) in dioxane (1.5 mL) was added HCl (5M aqueous) (3.5 mL, 17.5 mmol).
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The resulting mixture was sealed in a microwave vial and was brought to 90 °C and stirred for 16 h.
The mixture was concentrated under high vacuum on the rotory evaporator. The residue was purified
using preparatory HPLC (Method D) to afford 11 TFA salt (9.5 mg, 7 % yield) as a colorless oil. 1H
NMR (500 MHz, CD3OD) δ 7.48 (dd, J = 2.1, 0.4 Hz, 1H), 7.43 ꢀ 7.34 (m, 1H), 7.21 ꢀ 7.10 (m, 2H),
6.50 (dd, J =2.0, 0.5 Hz, 1H), 4.41 (dd, J = 12.6, 3.1 Hz, 1H), 3.71 (s, 3H), 3.22 (dd, J =14.8, 3.2 Hz,
1H), 2.53 (dd, J = 14.8, 12.7 Hz, 1H), 1.87 (s, 3H). MS (M+H)+: 323.0.
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(4S,6S)-4-(2,4-Difluorophenyl)-4-methyl-6-(pyrimidin-5-yl)-5,6-dihydro-4H-1,3-thiazin-2-
amine (12). To a solution of 18 (105 mg, 0.24 mmol) in dioxane (4 mL) was added 6.0 M HCl (4 mL,
24 mmol) at rt. The mixture was stirred at 95 °C for 1.5 h and at 55 °C for 18 h. The mixture was conꢀ
centrated. The crude material was purified via preparative HPLC (Method C) to give 12 (32 mg, 42%).
1H NMR (500 MHz, DMSOꢀd6)
9.10 (s, 1H), 8.85 (s, 2H), 7.94 ꢀ 7.60 (m, 1H), 7.18 ꢀ 6.81 (m, 2H),
4.79 (d, J = 8.9 Hz, 1H), 2.47 (br. s., 1H), 1.77 (d, J =11.3 Hz, 1H), 1.51 (s, 3H). MS (M+H)+: 321.2.
The absolute stereochemistry of this compound was confirmed by its coꢀcrystal structure with BACE1
active site.
(R,E)-N-(1-(2,4-Difluorophenyl)ethylidene)-2-methylpropane-2-sulfinamide (13). To a soꢀ
lution of 1ꢀ(2,4ꢀdifluorophenyl)ethanone (5 g, 32.0 mmol) in THF (80 mL) was added Rꢀ(+)ꢀtertꢀ
butansulfinamide (5.05 g, 41.6 mmol) and ethyl orthotitanate (14.5 mL, 70.5 mmol), and the resulting
mixture was heated at 65 °C for 12 h. The reaction was quenched by the addition of water (200 mL),
and the resulting mixture was filtered through Celite. The filtrate was then extracted with ethyl acetate
(3 x 50 mL). The combined extracts were washed with brine (50 mL), dried over MgSO , filtered and
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concentrated in vacuo. The crude material was purified by silica gel chromatography eluting with 30ꢀ
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70% ethyl acetate/hexanes to give 13 (3.1 g, 37 % yield). H NMR (500 MHz, CDCl3) δ 7.85 ꢀ 7.59
(m, 1H), 7.01 ꢀ 6.75 (m, 2H), 2.77 (d, J =2.7 Hz, 3H), 1.39 ꢀ 1.24 (s, 9H). MS (M+H)+: 260.05.
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(R)-N-((S)-2-(2,4-Difluorophenyl)-4-oxo-4-(pyrimidin-5-yl)butan-2-yl)-2-methylpropane-2-
sulfinamide (15). To a solution of diisopropylamine (0.90 mL, 6.3 mmol) in THF (8 mL) was added a
solution of 2.5 M nꢀBuLi in hexanes (2.8 mL, 6.9 mmol) at ꢀ78 °C under nitrogen. The mixture was
stirred from ꢀ78 °C to 0 °C over 30 min. This LDA solution was slowly added to a solution of 1ꢀ
(pyrimidinꢀ5ꢀyl)ethanone (733 mg, 6.00 mmol) in THF (10 mL) at ꢀ78 °C. After 30 min at ꢀ78 °C, a
solution of 13 (778 mg, 3.0 mmol) in 1 mL of THF was added slowly. The mixture was stirred from ꢀ78
°C to ꢀ30 °C over 5 h. Water was added, and the solution was extracted with 2 x150 mL of ethyl aceꢀ
tate. The combined extracts were dried over sodium sulfate and then filtered. The filtrate was evapoꢀ
rated in vacuo, and the residue was purified via silica gel chromatography eluting with 0ꢀ100% ethyl
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acetate/hexanes to give 15 (500 mg, 44 % yield). H NMR (500 MHz, CDCl3) δ 9.31 (s, 1H), 9.14 (s,
2H), 7.56 (td, J =9.2, 6.4 Hz, 1H), 6.97 ꢀ 6.85 (m, 1H), 6.69 (ddd, J =12.9, 8.5, 2.6 Hz, 1H), 5.28 (s,
1H), 4.17 (dd, J = 18.6, 1.2 Hz, 1H), 3.86 (dd, J = 18.7, 2.8 Hz, 1H), 1.82 (s, 3H), 1.29 (s, 9H). MS
(M+H)+: 382.05.
(R)-N-((2S,4R)-2-(2,4-Difluorophenyl)-4-hydroxy-4-(pyrimidin-5-yl)butan-2-yl)-2-
methylpropane-2-sulfinamide (16). To a solution of 15 (387 mg, 1.0 mmol) in ether (3 mL) was addꢀ
ed 1.0 M lithium triꢀtertꢀbutoxyaluminum hydride in THF (2 mL, 2 mmol) at ꢀ78 °C under nitrogen.
The mixture was stirred from ꢀ78 °C to ꢀ50 °C for 1 h. The reaction was quenched with Na2SO4.10H2O,
and 150 mL of ethyl acetate was added. After stirring at rt for 2 h, the solid was removed by filtration.
The filtrate was concentrated, and the residue was purified via silica gel chromatography elutong with 0ꢀ
10% methanol/ethyl acetate to give 16 (360 mg, 93 % yield) as a white foam. 1H NMR (500 MHz,
CDCl3) δ 9.06 (s, 1H), 8.72 (s, 2H), 7.42 (td, J =9.0, 6.4 Hz, 1H), 6.88 ꢀ 6.80 (m, 1H), 6.72 (ddd, J =
12.4, 8.7, 2.7 Hz, 1H), 6.58 (dd, J =3.7, 1.1 Hz, 1H), 6.10 (s, 1H), 5.25 (d, J =10.4 Hz, 1H), 2.65 (dd, J
= 14.8, 10.7 Hz, 1H), 2.11 (s, 3H), 1.93 (dd, J =14.8, 1.1 Hz, 1H), 1.22 (s, 9H). MS (M+H)+: 384.16.
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(1R,3S)-3-Amino-3-(2,4-difluorophenyl)-1-(pyrimidin-5-yl)butan-1-ol hydrochloride (17).
To a solution of 16 (360 mg, 0.94 mmol) in MeOH (3 mL) was added 4.0 M HCl in dioxane (3 mL, 12.0
mmol) at rt. The mixture was stirred at rt for 1 h. The mixture was concentrated and azeotroped with
toluene to give crude 17 hydrochloride (296 mg,100 % yield). This material was used for the next step
without any purification. A small portion of this material was basified with saturated sodium bicarꢀ
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bonate and extracted with ethyl acetate to give 17 (free base). H NMR (500 MHz, CDCl3) δ 9.01 (s,
1H), 8.69 (s, 2H), 7.25 (td, J = 9.2, 6.3 Hz, 1H), 6.86 ꢀ 6.69 (m, 2H), 5.17 (dd, J = 10.4, 2.9 Hz, 1H),
2.08 ꢀ 1.86 (m, 2H), 1.73 (s, 3H). MS (M+H)+: 280.05.
N-(((2S,4R)-2-(2,4-Difluorophenyl)-4-hydroxy-4-(pyrimidin-5-yl)butan-2-
yl)carbamothioyl)benzamide (18). To a solution of crude 17 hydrochloride (140 mg, 0.44 mmol) in
DCM (3 mL) was added DIPEA (0.31 mL, 1.77 mmol) followed by benzoyl isothiocyanate (80 mg,
0.49 mmol) at rt. The mixture was stirred at rt for 1.5 h. The solvents were removed in vacuo, and the
residue was was purified via silica gel chromatography 0ꢀ100% ethyl acetate/hexanes to give 18 (105
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mg, 54 % yield) as a colorless oil. H NMR (500 MHz, CDCl3) δ 11.75 (s, 1H), 9.07 (s, 1H), 8.71 (s,
2H), 7.92 ꢀ 7.79 (m, 2H), 7.66 ꢀ 7.60 (m, 1H), 7.54 ꢀ 7.47 (m, 2H), 7.43 ꢀ 7.34 (m, 1H), 6.91 ꢀ 6.85 (m,
1H), 6.76 (ddd, J =12.2, 8.7, 2.6 Hz, 1H), 5.05 (d, J =7.8 Hz, 1H), 3.67 (d, J = 2.6 Hz, 1H), 2.93 (dd, J
= 14.8, 9.6 Hz, 1H), 2.24 (3H, s), 2.21 (dd, J =14.8, 2.4 Hz, 1H). MS (M+H)+: 443.05.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
Xꢀray data summary of compounds 5 and 12, and methods for in vitro, in vivo, and pharmacokinetic asꢀ
says.
Molecular formula strings (cvs).
Accession Codes
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Structure coordinates of 5KR8 (5) and RKQF (12) have been deposited with the RSCB Protein Data
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Bank. Authors will release the atomic coordinates and experimental data upon article publication.
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AUTHOR INFORMATION
Corresponding Author
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* Tel: 203ꢀ677ꢀ7485. Eꢀmail: Yongꢀjin.wu@bms.com
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. Nicholas Meanwell for critically reviewing this manuscript. We also thanks Rudy Krause,
Alan Xu Lin, and Tracey Hall for various biological evaluations.
ABBREVIATIONS USED
TFA, trifluoroacetic acid; rt, room temperature; DCM, dichloromethane; Metstab, metabolic stability;
CYP, cytochrome P450 enzymes; PO, oral administration; IV, intravenous administration; PK/PD,
Pharmacokinetic/Pharmacodynamic; AUC, area under curve; B/P, brain/plasma concentration ratio.
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(10) (a) Oehlrich, D.; Prokopcova, H.; Gijsen, H .J. M. The evolution of amidineꢀbased brain peneꢀ
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C. R.; Nakano, M.; Jhee, S. S.; Yen, M.; Ereshefsky, L.; Lindstrom, T. D.; Calligaro, D. O.; Cocke,
P. J.; Hall, D. G.; Friedrich, S.; Citron, M.; Audia, J. E. Robust central reduction of amyloidꢀβ in
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brain Aβ reduction in rodents. ACS Med. Chem. Lett. 2016, 7, 271ꢀ276.
(14) Structure coordinates have been deposited with the RSCB Protein Data Bank (PDB ID 5KR8).
For details on crystallization and structure determination see the Supplemental Section.
(15) For an example of applying hydrogenꢀbond interactions involving 3,5ꢀdimethylisoxazole in drug
discovery, see: Hewings, D. S.; Wang, M.; Philpott, M.; Fedorov, O; Uttarkar, S.; Filippakopouꢀ
los, P; Picaud, S.; Vuppusetty, C.; Marsden, B.; Knapp, S.; Conway, S. J.; Heightman, T. D. . 3,5ꢀ
Dimethylisoxazoles act as acetylꢀlysineꢀmimetic bromodomain ligands J. Med. Chem. 2011, 54,
6761ꢀ6770.
(16) Structure coordinates have been deposited with the RSCB Protein Data Bank (PDB ID 5KQF).
For details on crystallization and structure determination see the Supplemental Section.
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(17) For human cytochrome P450 inhibition assay conditions, see: Wu, Y. ꢀJ; He, H.; Hu, S.; Huang,
Y.; Scola, P. M.; GrantꢀYoung, K.; Bertekap, R. L.; Wu, D.; Gao, Q.; Li, Y, Klakouski, C.; Westꢀ
phal, R. S. Identification of a potent and selective 5ꢀHT6 antagonist:ꢁ oneꢀstep synthesis of (E)ꢀ3ꢀ
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(benzenesulfonyl)ꢀ2ꢀ(methylsulfanyl)pyrido[1,2ꢀa]pyrimidinꢀ4ꢀylidenamine
from
2ꢀ
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(benzenesulfonyl)ꢀ3,3ꢀ bis(methylsulfanyl)acrylonitrile. J. Med. Chem. 2003, 46, 4834ꢀ4837.
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