Targeting the BACE1 Active Site Flap Leads to a Potent Inhibitor That Elicits Robust Brain Aβ Reduction in Rodents

Article abstract of DOI:10.1021/acsmedchemlett.5b00432

By targeting the flap backbone of the BACE1 active site, we discovered 6-dimethylisoxazole-substituted biaryl aminothiazine 18 with 34-fold improved BACE1 inhibitory activity over the lead compound 1. The cocrystal structure of 18 bound to the active site indicated two hydrogen-bond interactions between the dimethylisoxazole and threonine 72 and glutamine 73 of the flap. Incorporation of the dimethylisoxazole substitution onto the related aminothiazine carboxamide series led to pyrazine-carboxamide 26 as a very potent BACE1 inhibitor (IC₅₀ < 1 nM). This compound demonstrated robust brain Aβ reduction in rat dose-response studies. Thus, compound 26 may be useful in testing the amyloid hypothesis of Alzheimer's disease.

Full text of DOI:10.1021/acsmedchemlett.5b00432

Letter
pubs.acs.org/acsmedchemlett
Targeting the BACE1 Active Site Flap Leads to a Potent Inhibitor That
Elicits Robust Brain Aβ Reduction in Rodents
Yong-Jin Wu,*,† Jason Guernon,† Fukang Yang,† Lawrence Snyder,† Jianliang Shi,† Andrea Mcclure,†
Ramkumar Rajamani,† Hyunsoo Park,† Alicia Ng,† Hal Lewis,‡ ChiehYing Chang,‡ Dan Camac,‡
Jeremy H. Toyn,† Michael K. Ahlijanian,† Charles F. Albright,† John E. Macor,‡ and Lorin A. Thompson†
†Research and Development, Bristol-Myers Squibb Company, 5 Research Parkway, Wallingford, Connecticut 06492-7660, United
States
‡Research and Development, Bristol-Myers Squibb Company, PO Box 4000, Princeton, New Jersey 08543-4000, United States
S
* Supporting Information
ABSTRACT: By targeting the flap backbone of the BACE1 active site, we discovered 6-dimethylisoxazole-substituted biaryl
aminothiazine 18 with 34-fold improved BACE1 inhibitory activity over the lead compound 1. The cocrystal structure of 18
bound to the active site indicated two hydrogen-bond interactions between the dimethylisoxazole and threonine 72 and
glutamine 73 of the flap. Incorporation of the dimethylisoxazole substitution onto the related aminothiazine carboxamide series
led to pyrazine-carboxamide 26 as a very potent BACE1 inhibitor (IC50 < 1 nM). This compound demonstrated robust brain Aβ
reduction in rat dose−response studies. Thus, compound 26 may be useful in testing the amyloid hypothesis of Alzheimer’s
disease.
KEYWORDS: BACE1, inhibitor, aminothiazine, Aβ42, amyloid hypothesis, Alzheimer’s disease
ultiple lines of evidence suggest that β-amyloid (Aβ)
Mpeptide, particularly the longer 42 amino acid form Aβ42,
plays a critical role in the progression of Alzheimer’s disease
(AD).1−3 Aβ is derived from the β-amyloid precursor protein
(APP) by proteolysis. Human genetic mutations such as the
Swedish mutation result in increased β-secretase processing of
APP, elevated Aβ levels, and ultimately aggressive early onset
AD.4 A more recent study reported a variant of the APP gene
(APPA673T) at a site proximal to the BACE1 proteolytic site
that confers protection against AD.5 A673T substitution in APP
results in reduced production of Aβ peptides secreted from
heterologously transfected cells, further supporting the hypoth-
esis that increases in Aβ may underlie the pathology of AD.
Cleavage of APP by β-site APP cleaving enzyme-1 (BACE1)
results in shedding of the APP ectodomain, and the remaining
membrane bound C-terminal fragment, C99, is further processed
by γ-secretase to produce Aβ. Thus, inhibition of BACE1 to
reduce Aβ production is a promising approach to test the
amyloid hypothesis.6−10
In 2009, Eli Lilly reported that 1 (LY2811376) (Figure 1), a
small molecule biaryl aminothiazine BACE1 inhibitor, demon-
strated sustained brain Aβ reduction in healthy volunteers upon
oral administration.11,12 The clinical development of this
compound was discontinued due to nonclinical, off-target-
related retinal toxicity. As this compound exhibited only modest
Figure 1. Structure of 1 (LY2811376).
BACE1 inhibitory activity (IC50: 0.27 μM from our assay, 0.24
μM from the literature12), we sought to increase its potency as an
avenue to increase the therapeutic window versus the observed
toxicity. To our knowledge, no potent biaryl aminothiazine
BACE1 inhibitors have been disclosed. We recently developed a
novel approach to improve the activity of the biarylaminothia-
zines, and we then extended this strategy to the related
aminothiazine carboxamide series. These efforts culminated in
the discovery of a potent BACE1 inhibitor that elicited robust
brain Aβ reduction in rodents. This report describes our progress
along these lines.
Received: November 12, 2015
Accepted: January 11, 2016
© XXXX American Chemical Society
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Examination of the docked conformation of 1 to the BACE1
active site revealed proximity between threonine 72 and
glutamine 73 of the flap and the C-6 position of the thiazine
ring. We hypothesized that an appropriately selected substituent
at C-6 may interact with the flap residues, thus enhancing binding
affinity (Figure 2). Unfortunately, the existing synthesis of the 6-
4,6-cis products were predominant over the trans isomers. The
minor trans isomers were significantly more active than their
corresponding cis counterparts (data not shown). The most
active compound from this small library was the (4S,6S)-6-(3,4-
dimethoxyphenyl)-substituted aminothiazine 7 (IC50: 1.1 μM,
Scheme 2). We then introduced the difluoro substituents onto 7
using 2,4-difluoro-5-(pyrimidin-5-yl)-benzaldehyde (4b,
Scheme 2) in the three-component reaction, and the resulting
analogue 8 exhibited an approximately 4-fold improved BACE1
inhibitory activity (IC50: 0.29 μM). This magnitude of
improvement is consistent with that observed in the 6-
unsunstituted biaryl aminothiazine series previously reported.12
Our next library synthesis was aimed at enhancing potency,
reducing lipophilicity (clogP: 3.6 for 8 versus 2.9 for 1), and
improving ligand efficiency (LE: 0.29 kcal·mol−1 for 8 versus 0.37
kcal·mol−1 for 1). Molecular modeling of 8 indicated the
potential for hydrogen-bond interactions between the two
methoxy groups and the flap backbone, and these interactions
may be improved by moving the oxygen into the ring, i.e.,
forming a heterocyclic ring system. Replacement of dimethox-
yphenyl with an appropriate heterocycle was also anticipated to
reduce lipophilicity and improve ligand efficiency. Thus, three-
component reactions of vinyl heterocycles with 3-(pyrimidin-5-
yl)-benzaldehyde (4a) were carried out, and this exercise led to
6-(3,5-dimethylisoxazol-4-yl) substituted trans aminothiazine
( )-9 with moderate activity (IC50: 0.23 μM, Figure 3). Again,
Figure 2. Modeled interaction between the flap backbone of the BACE1
active site and a substituent at the C-6 of aminothiazine.
substituted aminothiazines 2 was both lengthy and linear, and
therefore we desired to develop a more convergent method-
ology. Retrosynthetically, aminothiazines 2 could be assembled
through a [4 + 2] cycloaddition involving heterodiene 3 and an
olefin, and the heterodiene 3 can be visualized from the
condensation of an aryl aldehyde or ketone and thiourea
(Scheme 1). Indeed, the three-component, one-pot reaction was
Scheme 1. Retrosynthesis of 6-Substituted Aminothiazines
previously described for the synthesis of 4-desmethyl amino-
thiazines,13 and with some minor modifications, we were able to
obtain a variety of 4-des-methyl 6-substituted aminothiazines (2,
R′ = H). However, we were unable to extend this methodology to
the synthesis of 4-methyl 6-substituted analogues (2, R′ = Me).
At the time this work was carried out (January 2011), the impact
of this methyl group was unknown in the literature.14 Therefore,
our plan was to identify an active 4-desmethyl, 6-substituted
aminothiazine using the [4 + 2] cycloaddition strategy and then
to evaluate the impact of the 4-methyl group on potency.
Our work began with the three-component, one-pot reaction
of the commercially available 3-(pyrimidin-5-yl)-benzaldehyde
(4a) with a variety of substituted styrenes 5 (Scheme 2). In
general, these reactions proceeded in moderate yields, and the
Figure 3. Structures of ( )-9, 10, ( )-11, and ( )-12.
we introduced the difluoro substituents, and the resulting
(4S,6S) enantiomer 10 exhibited activity 5-fold more potent than
the lead compound 1. As compared to the dimethoxyphenyl
analogue 8, compound 10 also showed reduced lipophilicity
(clogP: 2.9 versus 3.6 for 8) and improved ligand efficiency (LE:
0.35 kcal·mol−1 versus 0.29 kcal·mol−1 for 8). As observed in
other pairs of enantiomers, the opposite enantiomer of 10 was
significantly less active (data not shown). Introduction of a
quaternary methyl at C-6 in ( )-11 dramatically reduced activity,
while the activity was restored upon removal of the C-3 methyl of
the isoxazole in ( )-12 (vide infra).
To better understand the binding conformation of the
dimethylisoxazole group, we obtained a cocrystal structure of
thiazine 10 bound in the BACE1 active site (Figure 4).15 As
expected, the aminothiazine functionality formed intricate
hydrogen-bond donor−acceptor interactions with aspartates 32
and 228 at the BACE1 active site; the C-2 hydrogen (see 10 in
Figure 3) attached to the difluorophenyl ring formed a potential
C−H hydrogen-bond with the backbone carbonyl of glycine 230;
and the pyrimidine ring was projected into the opening of S3
pocket where it interacted with the carbonyl of serine 229 via a
bridging water molecule. The two heteroatoms of the isoxazole
ring served as hydrogen-bond acceptors to the amide NH groups
of the threonine 72 and glutamine 73 located on the flap
Scheme 2. Synthesis of 6-Substituted Aminothiazines
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a
Scheme 3. Synthesis of Aminothiazine 18
Figure 4. Cocrystal structure of 10 bound with BACE1 active site, with
electron density contoured around 10 in blue mesh.15
backbone.16 The C-6 isoxazole ring was arranged nearly
perpendicularly to the thiazine ring, and introduction of a
quaternary methyl at C-6 as in compound 11 would cause severe
steric repulsion between the two methyl groups (Figure 5). As a
a
Reagents and conditions: (a) benzoyl isothiocyanate, CH2Cl2, rt,
94%; (b) 6 N HCl, dioxane, 100 °C, 6 h, 49%; (c) Boc2O, saturated
NaHCO3, 1,4-dioxane, water, rt, 75%; (d) pyrimidin-5-yl-boronic acid,
PdCl2(PPh3)2, Cs2CO3, DME, EtOH, H2O, 100 °C, 5 min, 60%; (e)
TFA, CH2Cl2, rt, 100%. The thermal ellipsoid plot (50% ellipsoids) of
16 is also shown above.
Figure 5. Steric interaction of the C-6 methyl of the thiazine ring with
the C-3 methyl of isoxazole in 11.
result, the orthogonal arrangement would be unfavorable, thus
resulting in diminished activity (( )-11 IC50: 3.7 μM). However,
when the C-3 methyl of the isoxazole was removed, as in
compound 12, activity was restored. Indeed, the active
enantiomer of compound ( )-12 was estimated to have an
IC50 value of 90 nM (only one enantiomer was active based on
SAR), comparable to that obtained with compound 10 (IC50: 70
nM, Figure 3).
With the C-4 des-methyl thiazine 10 identified as a moderately
potent BACE1 inhibitor, our next step was to prepare the C-4
methyl-substituted aminothiazine 18 (Scheme 3). Reaction of
amino alcohol 13, readily available using methodology recently
developed in our laboratory,17 with benzoyl isothiocyanate
provided the benzoyl thiourea derivative 14. Treatment of 14
with hydrochloric acid at 100 °C brought about ring closure and
subsequent hydrolysis of the benzoyl group to give the 4,6-trans-
aminothiazine 15, which was converted to its NHBoc derivative
16. The relative and absolute stereochemistry of 16 were
confirmed by single crystal X-ray crystallography (Scheme 3).18
Sukuki coupling of 16 with pyrimidin-5-yl-boronic acid and
subsequent N-Boc deprotection furnished 18.
Figure 6. Overlay of cocrystal structures of 10 (violet) and 18 (yellow)
bound with BACE1 active site.
group led to a loss of this energy barrier to access the preferred
pseudoaxial conformation (Figure 7). In the case of compounds
10 and 18, quantum mechanical calculations at the HF/6-31+G*
level of theory showed that both compounds highly preferred the
bound pseudoaxial conformations, by 10.2 kcal/mol for 10 and
5.3 kcal/mol for 18. We conclude that the 4-methyl group of 18
Compound 18 (IC50: 8 nM) was nearly 10-fold more potent
than the 4-des-methyl analogue 10. A similar trend was also
observed in the iminopyrimidinone BACE1 inhibitors.19 Again,
we obtained a cocrystal structure of 18 in the BACE1 active
site,20 and Figure 6 shows the overlay of cocrystal structures of
both 10 and 18. The biaryl group in both inhibitors is oriented in
a pseudoaxial conformation to occupy the contiguous S1−S3
hydrophobic pockets. A similar effect was seen in the related
iminopyrimidinone analogue;19 however, we believe the under-
lying energetics are different. In the iminopyrimidinone case, the
pseudoequatorial conformation was preferred by 1.4 kcal/mol
with a C-4 hydrogen substituent; addition of the C-4 methyl
Figure 7. Energy difference between pseudoaxial and pseudoequatorial
conformations of 10 and 18.
C
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contributes to activity primarily through a favorable hydrophobic
interaction with isoleucine 118, which lines the active site instead
of conformational preference as shown in the iminopyrimidi-
none analogues.
Despite its potent activity, compound 18 demonstrated only
modest reduction in brain Aβ peptides in rodents relative to the
vehicle treated controls even at 30 mg/kg po, a result of moderate
brain penetration. The exposure was limited by poor metabolic
stability across species: only 23% (human), 49% (rat), and 48%
(mouse) remained after a 10 min incubation with liver
microsomes at 0.5 μM. The major metabolites resulted from
oxidation of the pyrimidine ring, a pathway difficult to address
because BACE1 inhibitory activity was sensitive to substitution
of this ring. In the end, we were unable to improve metabolic
stability while maintaining potent BACE1 inhibitory activity.
While our work to increase the potency of biaryl amino-
thiazines was being undertaken, workers at Eli Lilly also disclosed
the discovery of furo[3,4-d][1,3]thiazin-2-amine picolinamide
19 (LY2886721) (Figure 8), a potent BACE1 inhibitor that
Figure 9. Docking conformation of 22 bound in the BACE1 active site.
Indeed, the 5,6-difluorophenyl analogue 23 was 44-fold more
potent than 22 with an IC50 value of less than 1 nM. The superior
activity of the 5,6-difluorophenyl picolinamides over their 4,6-
difluorophenyl counterparts appeared to be general, but the
magnitude of the enhancement depended on the substituent(s)
of the picolinic acid moiety. The 5,6-difluorophenyl picolina-
mides displayed either comparable or slightly improved activity
over the 5-fluorophenyl analogues (data not shown). As we had
expected, fluoro substitution at C-2 of the phenyl ring was not
well tolerated (25, IC50: 160 nM) as this eradicated a potential
C−H hydrogen-bond between the C-2 hydrogen of the phenyl
and the carbonyl of Gly230 in the BACE1 backbone, as shown in
the cocrystal structure of biaryl aminothiazine 10 (Figure 4).
Overall, given a specific picolinamide, the activity of the
analogues can be arranged in the order of 5,6-difluorophenyl
(23) ≥ 5-fluorophenyl (24) > 4,6-difluorophenyl (22) > 2-
fluorophenyl (25).
Figure 8. Structures of 19 (LY2886721) and 20.
reached phase II clinical trials in mild cognitive impairment.21,22
This new disclosure from Eli Lilly prompted us to replace the
pyrimidyl moiety of 18 with a picolinamide. To test this
approach, we prepared the 4-des-methyl aminothiazine 20 using
the three-component, one-pot reaction of 5-chloro-N-(4-fluoro-
3-formylphenyl)picolinamide, 3,5-dimethyl-4-vinylisoxazole and
thiourea (scheme not shown). Compound 20 displayed good
BACE1 inhibitory activity (IC50: 5 nM). As the C-4 quaternary
methyl of the thiazine ring and the C-4 fluoro substituent of the
phenyl ring generally contributed to the activity of the biaryl
aminothiazine BACE1 inhibitors, we prepared picolinamide 22
from bromide 16 in four steps: palladium-catalyzed azidation,
reduction of azide to aniline 21, amidation of the aniline, and
removal of the N-Boc group (Scheme 4). Interestingly,
The fluoro substituents exerted a profound impact not only on
BACE1 inhibitory activity of the aminothiazine picolinamides,
but also on their metabolic stability (Figure 10). For example, C-
a
Scheme 4. Synthesis of Aminothiazine 24
a
Figure 10. Metstab (H/R/M): % percent compound that remained
after a 10 min incubation with (H)uman, (R)at, or (M)ouse liver
microsomes at 0.5 μM.
Reagents and conditions: (a) NaN3, CuSO4·5H2O, L-ascorbic acid
sodium salt, trans-1,2-(bismethylamino)cyclohexane, 80 °C; (b) Pd/C
(10%), MeOH, H2 balloon, rt, 37% for two steps; (c) 5-chloropicolinic
acid, HATU, Hunig’s base, CH2Cl2, rt, 80%; (d) TFA, 61%
2 fluoro substitution (25) was detrimental to both activity and
metabolic stability. Our structure−metabolic stability relation-
ship studies revealed the following order of metabolic stability:
5,6-difluorophenyl (23), 4,6-difluorophenyl (22) > 2-fluoro-
phenyl (25), 5-fluorophenyl (24). Taken together, we concluded
that the 5,6-difluorophenyl picolinamides provided the optimal
balance between BACE1 inhibitory activity and metabolic
stability. Accordingly, we focused on 5,6-difluorophenyl based
carboxamides. Optimization of the amide side chain in the S3
pocket culminated in the discovery of pyrazine-carboxamide 26
compound 22 was 6-fold less active than the 4-desmethyl
analogue 20. The docking conformation of 22 (Figure 9) in the
BACE1 active site indicated that the NH of the amide formed a
hydrogen-bond with the backbone carbonyl of Gly230. The
electronic repulsion between the 2-fluorine substituent on the
phenyl and the amide carbonyl resulted in a nonoptimal
hydrogen-bond configuration for the backbone carbonyl of
Gly230. Relocation of fluoro substituent from C-4 to C-5 of the
phenyl was expected to minimize this electronic repulsion.
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(Scheme 5) as a potent BACE1 inhibitor (IC50: 0.9 nM) with a
good metabolic stability profile. Compound 26 was prepared
a
Scheme 5. Synthesis of Aminothiazine 26
Figure 12. Brain Aβ42 reduction and plasma concentration curve of 26.
a
Reagents and conditions: (a) 5-methoxypyrazine-2-carboxylic acid,
IC50 (1 nM) implied significant brain drug binding for
compound 26.
HATU, Hunig’s base, CH2Cl2; (b) TFA, CH2Cl2, 63% for two steps.
Compound 26 showed an IC50 of 0.64 μM in the hERG whole-
cell patch clamp assay, which provided a 49-fold separation
versus the brain Aβ42 EC50 of 13 nM free drug level in plasma.
More studies would be required to determine if hERG inhibition
is a potential safety issue for this compound.
The pyrazine-carboxamide 26 contains an amide bond, which
could potentially be hydrolyzed in vivo to release the free aniline
28 (Figure 13). Indeed, in the mouse Aβ reduction studies,
from aniline 27, which was available following the exact sequence
described for compound 21 (Scheme 4).
Compound 26 demonstrated robust brain Aβ reduction in
rodents. A single oral administration of 26 at 10 mg/kg in mice
resulted in a reduction of brain Aβ42 peptide relative to vehicle-
treated control by 85% (data not shown). In rat studies, this
compound demonstrated a dose-dependent reduction of brain
Aβ40, Aβ42, and total Aβ (Aβ1‑x) when dosed orally (Figure 11).
Figure 13. Thermal ellipsoid plot (50% ellipsoids) of aniline 28.
aniline 28 was detected in the plasma 3 h after oral
administration. Free anilines per se are not inherently mutagenic,
but further metabolism generates species that could be
mutagenic. To address this concern, we submitted 28 to the
standard Ames test, in which it was determined to be
nonmutagenic. Compound 28 was obtained from 27 (Scheme
5) via removal of the N-Boc group. The absolute configuration of
28 was confirmed by way of single-crystal X-ray diffraction
analysis (Figure 13).23
The cytochrome P450 inhibitory potential of compound 26
was also determined using recombinant CYP1A2, CYP2C9,
CYP2C19, CYP2D6, and CYP3A4 in order to assess the
potential likelihood of drug−drug interactions. Low levels of
inhibition were observed at all major human P450 isozymes
(IC50 > 20 μM) with the highest level of inhibition against
CYP2C8 (IC50: 5 μM).
Figure 11. Dose-dependent inhibition of Aβ40, Aβ42, and total Aβ
(Aβ1−x) in rat brain by 28 at 5 h after oral administration (n = 5 rats).
The exposure of 26 was also measured in rat plasma and brain
samples (Table 1) to allow quantification of the PK/PD
Table 1. Brain, Plasma Concentration, and Brain/Plasma
a
Ratio of 26
dose
(mg/kg)
plasma conc (nM)
(SD)
brain conc (nM)
(SD)
brain/
plasma
1
150 (65)
360 (75)
1500 (320)
67 (46)
0.44 (0.14)
0.46 (0.10)
0.30 (0.10)
3
160 (30)
420 (190)
10
a
Compound dosed PO to female rats in PEG-400/EtOH/Tween 80
In summary, we recognized certain features in 1 and used them
to develop a general approach to enhance its potency by targeting
the flap backbone in the active site of BACE1. This approach led
to dimethoxyphenyl at C-6 of the thiazine ring as an initial binder,
which incorporated weak hydrogen-bond acceptors to the flap
backbone. The interactions with the flap were optimized with the
dimethylisoxazole heterocycle. The quaternary methyl substitu-
ent at C-4 was introduced, and the resulting aminothiazine 18
was 34-fold more potent than 1. We then replaced the pyrimidyl
moiety with a carboxamide, and our SAR studies showed that the
5,6-difluorophenyl moiety in the S1 pocket was optimal in terms
of activity and metabolic stability. Optimization of the amide side
chain in the S3 pocket culminated in the discovery of the
pyrazine-carboxamide 26 as a potent BACE1 inhibitor with good
metabolic stability. This compound demonstrated robust brain
Aβ lowering after oral dosing in rat dose−response studies.
(84:15:1); plasma and brain collected 5 h postdose (5 rats/dose) for
determination of exposure and brain Aβ levels. Data are mean values
(n = 5) with SD in parentheses.
relationship (Figure 12). Analysis of the rat brain Aβ42 reduction
versus the total plasma concentration curve (Figure 12) resulted
in the estimation of a brain Aβ42 EC50 of 260 nM total plasma
concentration or 13 nM free drug plasma level (using rat plasma
protein binding data for 28, 95%), which is 14-fold the cellular
IC50 value of the compound. In terms of brain exposure required
for efficacy, 260 nM total plasma exposure was achieved close to
the 3 mg/kg drug dose (Table 1), which gave 360 nM plasma and
160 nM total brain exposure. Therefore, the total brain exposure
at the ED50 was near 160 nM. Comparing this value to the cell
E
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(9) Yan, R.; Vassar, R. Targeting the β secretase BACE1 for
Alzheimer’s disease therapy. Lancet Neurol. 2014, 13, 319.
Taken together, we believe that compound 26 can be considered
for further preclinical evaluations for testing the amyloid cascade
hypothesis.
(10) Oehlrich, D.; Prokopcova, H.; Gijsen, H. J. M. The evolution of
amidine-based brain penetrant BACE1 inhibitors. Bioorg. Med. Chem.
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(11) Audia, J. E.; Mergott, D. J.; Dustin, J.; Sheehan, S. M.; Watson, B.
M. Aminodihydrothiazine derivatives as BACE inhibitors for the
treatment of Alzheimer’s diseases. WO-2009/134617.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00432.
(12) May, P. C.; Dean, R. A.; Lowe, S. L.; Martenyi, F.; Sheehan, S. M.;
Boggs, L. N.; Monk, S. A.; Mathes, B. M.; Mergott, D. J.; Watson, B. M.;
Stout, S. L.; Timm, D. E.; LaBell, E. S.; Gonzales, 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 amyloid-β in humans with an orally available, non-
peptidic β-secretase inhibitor. J. Neurosci. 2011, 31, 16507−16516.
(13) Zhu, Y.; Huang, S.; Wan, J.; Yan, L.; Pan, Y.; Wu, A. Two novel
diastereoselective three-component reactions of alkenes or 3,4-dihydro-
(2H)-pyran with urea/thiourea-aldehyde mixtures: [4 + 2] cyclo-
addition vsbBiginelli-type reaction. Org. Lett. 2006, 8, 2599−2602.
(14) After this work was near completion, Lilly disclosed LY2811376
in ref 12 by the end of 2011. This paper suggests that the purpose of the
quaternary methyl group is to exacerbate the desired topology and
enhance chemical stability, but no details were provided.
Synthetic methods and characterization data for com-
pounds 6−28 and methods for in vitro, in vivo, and
pharmacokinetic assays (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: 203-677-7485. E-mail: yong-jin.wu@bms.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(15) Structure coordinates have been deposited with the RSCB Protein
Data Bank (PDB ID 5ENM).
We thank Dr. Nicholas Meanwell for critically reviewing this
manuscript. We also thank Rudy Krause, Alan Xu Lin, and Tracey
Hall for various biological evaluations, and A. Arumugam, K.
Mahammed, Murali Botlagunta, P. N. Arunachalam, Arun Kumar
Gupta, and Richard Rampulla for scaling up intermediate 15.
(16) For an example of applying hydrogen-bond interactions involving
3,5-dimethylisoxazole in drug discovery, see: Hewings, D. S.; Wang, M.;
Philpott, M.; Fedorov, O.; Uttarkar, S.; Panagis Filippakopoulos, P.;
Picaud, S.; Vuppusetty, C.; Marsden, B.; Knapp, S.; Conway, S. J.;
Heightman, T. D. 3,5-Dimethylisoxazoles act as acetyl-lysine-mimetic
bromodomain ligands. J. Med. Chem. 2011, 54, 6761−70.
(17) Guernon, J.; Marcin, L.; Higgins, M.; Yang, F.; Shi, J.; Snyder, L.;
Thompson, L. A.; Wu, Y.-J. Synthesis of β-disubstituted β-amino
isoxazolyl ketones by addition of ketimines with isoxazolyl methyl
ketone enolates. Tetrahedron Lett. 2014, 55, 2134−2137.
(18) The crystal structure of 16 has been deposited in the Cambridge
Crystallographic Data Centre (CCDC #: 1436245).
(19) Edwards, P. D.; Albert, J. S.; Sylvester, M.; Aharony, D.; Andisik,
D.; Callaghan, O.; Campbell, J. B.; Carr, R. A.; Chessari, G.; Congreve,
M.; Frederickson, M.; Folmer, R.H. A.; Geschwindner, S.; Koether, G.;
Kolmodin, K.; Krumrine, J.; Mauger, R. C.; Murray, C. W.; Olsson, L.;
Patel, S.; Spear, N.; Tian, G. Application of fragment-based lead
generation to the discovery of novel, cyclic amidine β-secretase
inhibitors with nanomolar potency, cellular activity, and high ligand
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(20) Structure coordinates have been deposited with the RSCB Protein
Data Bank (PDB ID 5ENK).
(21) May, P. C.; Willis, B. A.; Lowe, S. L.; Dean, R. A.; Monk, S. A.;
Cocke, P. J.; Audia, J. E.; Boggs, L. N.; Borders, A. R.; Brier, R. A.;
Calligaro, D. O.; Day, T. A.; Ereshefsky, L.; Erickson, J. A.; Gevorkyan,
H.; Gonzales, C. R.; James, D. E.; Jhee, S. S.; Komjathy, S. F.; Li, L.;
ABBREVIATIONS USED
■
TFA, trifluoroacetic acid; TEA, triethylamine; DMF, N,N-
dimethylformamide; HATU, 1-[bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexa-
fluorophosphate; rt, room temperature; Metstab, metabolic
stability; CYP, cytochrome P450 enzymes; po, oral admin-
istration; PK/PD, pharmacokinetic/pharmacodynamic
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DOI: 10.1021/acsmedchemlett.5b00432
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX