Spirocyclic β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors: From hit to lowering of cerebrospinal fluid (CSF) amyloid β in a higher species

Article abstract of DOI:10.1021/jm4002154

A hallmark of Alzheimer's disease is the brain deposition of amyloid beta (Aβ), a peptide of 36-43 amino acids that is likely a primary driver of neurodegeneration. Aβ is produced by the sequential cleavage of APP by BACE1 and γ-secretase; therefore, inhibition of BACE1 represents an attractive therapeutic target to slow or prevent Alzheimer's disease. Herein we describe BACE1 inhibitors with limited molecular flexibility and molecular weight that decrease CSF Aβ in vivo, despite efflux. Starting with spirocycle 1a, we explore structure-activity relationships of core changes, P3 moieties, and Asp binding functional groups in order to optimize BACE1 affinity, cathepsin D selectivity, and blood-brain barrier (BBB) penetration. Using wild type guinea pig and rat, we demonstrate a PK/PD relationship between free drug concentrations in the brain and CSF Aβ lowering. Optimization of brain exposure led to the discovery of (R)-50 which reduced CSF Aβ in rodents and in monkey.

Full text of DOI:10.1021/jm4002154

Article
pubs.acs.org/jmc
Spirocyclic β‑Site Amyloid Precursor Protein Cleaving Enzyme 1
(BACE1) Inhibitors: From Hit to Lowering of Cerebrospinal Fluid (CSF)
Amyloid β in a Higher Species
Kevin W. Hunt,*,† Adam W. Cook,† Ryan J. Watts,‡ Christopher T. Clark,†,§ Guy Vigers,† Darin Smith,†
Andrew T. Metcalf,† Indrani W. Gunawardana,† Michael Burkard,† April A. Cox,†,∥ Mary K. Geck Do,†,⊥
Darrin Dutcher,†,# Allen A. Thomas,† Sumeet Rana,† Nicholas C. Kallan,†,∞ Robert K. DeLisle,†
James P. Rizzi,†,¶ Kelly Regal,†,× Douglas Sammond,†,○ Robert Groneberg,†,△ Michael Siu,‡
Hans Purkey,‡ Joseph P. Lyssikatos,‡ Allison Marlow,† Xingrong Liu,‡ and Tony P. Tang†
†Array BioPharma, 3200 Walnut Street, Boulder, Colorado 80301, United States
‡Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
ABSTRACT: A hallmark of Alzheimer’s disease is the brain deposition
of amyloid beta (Aβ), a peptide of 36−43 amino acids that is likely a
primary driver of neurodegeneration. Aβ is produced by the sequential
cleavage of APP by BACE1 and γ-secretase; therefore, inhibition of
BACE1 represents an attractive therapeutic target to slow or prevent
Alzheimer’s disease. Herein we describe BACE1 inhibitors with limited
molecular flexibility and molecular weight that decrease CSF Aβ in vivo,
despite efflux. Starting with spirocycle 1a, we explore structure−activity
relationships of core changes, P3 moieties, and Asp binding functional
groups in order to optimize BACE1 affinity, cathepsin D selectivity, and blood−brain barrier (BBB) penetration. Using wild type
guinea pig and rat, we demonstrate a PK/PD relationship between free drug concentrations in the brain and CSF Aβ lowering.
Optimization of brain exposure led to the discovery of (R)-50 which reduced CSF Aβ in rodents and in monkey.
the early stages of the disease. This view was bolstered by the
recent discovery of a coding mutation in the APP gene that
protects against AD and cognitive decline in the elderly. The
mutation is near the aspartyl protease β-site of APP and reduces
in vitro Aβ production by approximately 40%.7
The enzyme that initiates the cascade of Aβ production is the
β-site APP cleaving enzyme 1 (BACE1), also known as
membrane-associated aspartic protease 2 (memapsin 2) and
aspartyl protease 2 (ASP2).8 BACE1 is most highly expressed
in the brain where it cleaves APP to release the amino-terminal
fragment of APP (sAPPβ) and the membrane bound C-
terminal fragment C99. Cleavage of the C99 fragment by γ-
secretase releases Aβ.9
INTRODUCTION
■
Alzheimer’s disease (AD) is an irreversible neurodegenerative
disease that affects more than 18 million patients worldwide.
The cost of care for AD patients exceeds $172 billion in the
U.S. alone, despite over 15 million family members providing
unpaid care. By 2050, the U.S. cost of care is predicted to
exceed $1 trillion.1 The consequences of AD (principally the
projected stresses upon health care systems) have spurred
greater government funding for AD by France and the United
States.2 Further investment from government agencies is
timely, as many pharmaceutical companies are divesting CNS
research.3
Modifying the course of AD may be possible by decreasing
the production of amyloid β peptides (Aβ), the presumptive
molecules central to the amyloid hypothesis resulting from
metabolism of the amyloid precursor protein (APP).4 This
hypothesis is based upon the increased production of Aβ in
patients with dominantly inherited forms of AD, the high
Aβ1−40,42 content in plaques from post-mortem brains of AD
patients, and experiments demonstrating the neuronal toxicity
of Aβ. Further, Down syndrome patients, who often develop
AD pathology by age 50, have increased Aβ levels from birth
and show plaque deposition as early as their preteen years.5
The amyloid hypothesis has been modified slightly to focus on
the greater in vivo neurotoxicity of Aβ oligomers vs
monomers.6 A corollary to the amyloid hypothesis is that
decreasing Aβ production will be disease modifying, at least in
The therapeutic potential for BACE1 inhibition coupled with
the inferred safety from the viability of BACE1−/− mice10 has
resulted in numerous publications about the discovery and
characterization of BACE1 inhibitors.11 Recently, Eli Lilly
researchers published phase I clinical results for a BACE1
inhibitor yielding important proof of mechanism data for this
pathway in humans. While the Lilly compound effectively
lowered CSF Aβ levels, advancement of the molecule was
halted because of retinal pathology identified in a 3-month rat
toxicology study.12 The observed retinal pathology of the Lilly
compound is consistent with observations from animals
Received: February 13, 2013
© XXXX American Chemical Society
A
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deficient in the aspartyl protease cathepsin D (CatD).13 These
data suggest that selectivity for BACE1 over CatD inhibition is
essential.
a
Scheme 1. Synthesis of Aminohydantoins
The efficacy of a BACE1 inhibitor is equally dependent upon
blood−brain barrier (BBB) permeability as it is upon potency.
While protecting the brain from exposure to pollutants and
toxins, the BBB also limits the entry of potentially beneficial
xenobiotics such as pharmaceutical agents.14 Fortunately,
limiting total polar surface area (tPSA < 90),15 molecular
weight (MW < 450), lipophilicity (cLogP < 4), hydrogen bond
donors (<3), and rotatable bonds (molecular flexibility)
generally enhances free drug exposure to the brain.16
Adherence to these limits increases the probability of
successfully discovering CNS penetrant molecules.
A key advancement in BACE1 inhibitor design is the
discovery of 2-amino heterocycles that engage both active site
aspartyl moieties of BACE1.17 These ligands enable a better
balance of physical−chemical properties, improved brain
penetrance, and in vivo efficacy. Although highly potent, the
reported 2-amino heterocyclic inhibitors often utilize cores that
are highly aromatic or contain multiple rotatable bonds.
Seeking to identify novel, highly soluble, and permeable
BACE1 inhibitors, we prepared a collection of small, rigid,
and saturated or partially saturated cores utilizing 2-amino
heterocycles, leading to the identification of spirocycle
hydantoin 1a (BACE1 IC50 = 36.8 μM). Spirocycle 1a (Figure
1) presents an ideal core for optimization, as it combines (1)
a
Reagents and conditions. (a) For X = O, S: (NH4)2CO3, KCN,
DMA, 80 °C. For X = CH2: (NH4)2CO3, KCN, EtOH, 150 °C (steel
pressure bomb). (b) K2CO3, RX, DMF, rt; (c) Lawesson’s reagent,
t
toluene, reflux; (d) NH3, BuOOH, MeOH, H2O, rt; (e) R′B(OH)2,
Pd(PPh3)4, Na2CO3, dioxane, H2O, 80 °C; (f) BocNH2, Pd2(dba)3,
XPhos, Cs2CO3, THF, rt; (g) TFA, DCM, rt; (h) 5-methylfuran-2-
carbonyl chloride, Et3N, DCM, rt.
1a−c. A Suzuki reaction or amination−acylation sequence
provided analogues 11−50 and 53−56. Further diversity was
introduced onto the spiro-hydantoin-tetrahydronaphthalene
(THN) core by benzylic oxidation, benzylic fluorination, and
subsequent Suzuki coupling (Scheme 2).
Scheme 2. Synthesis of Tetrahydronaphthalone and
Difluoronaphthalene Analogues
a
a
t
Reagents and conditions: (a) CrO3, BuOOH, DCM, rt; (b) 5-
Figure 1. X-ray cocrystal structure of BACE1 and 1a. The surface of
the protein is colored by electrostatic potential. Part of the flexible
“flap” including Tyr14 is visible at the top of the image. The two key
active site aspartic acids, Asp32 and Asp228, are also labeled.
pyrimidinylboronic acid, PdCl2(dppf)·DCM, Na2CO3, dioxane, H2O,
80 °C; (c) bis(2-methoxyethyl)aminosulfur trifluoride, DCE, rt.
The aminooxazoline and aminothiazoline heterocycles were
readily introduced onto olefin 6, obtained by Tebbe olefination
of ketone 2c (Scheme 3). Treatment of 6 with iodine and
either silver cyanate or silver thiocyanate provided intermediate
limited molecular flexibility with three-dimensionality, key
factors for improved permeability and solubility,18,19 (2)
accessible vectors to the S3 and S2′ sites of BACE1, (3)
utilization of a 2-amino heterocycle to engage both Asp32 and
Asp228 of the BACE1 active site, and (4) low MW (338.2).
Note that only the density of the (R)-enantiomer of racemate
1a was detectable in the BACE1 active site.
Scheme 3. Synthesis of Aminooxazoline and
a
Aminothiazoline Analogues
CHEMISTRY
■
To limit the number of rotatable bonds while preserving three-
dimensionality, our investigation utilized spiro-bicyclic cores
that omit P2′ groups. The synthesis of aminohydantoin
analogues commenced with Bucherer−Bergs reaction of
ketones 2a−c20 (Scheme 1). Selective alkylation of the imide
nitrogen of the intermediate hydantoins provided alkylated
hydantoins 3a−c. Reaction with Lawesson’s reagent followed
by oxidative amination provided the desired spiro-bicyclic cores
a
Reagents and conditions: (a) Tebbe reagent, PhMe or DCM, rt. (b)
For XO: AgOCN, I2, THF, ether, 0 °C. (c) For XS: AgSCN, I2,
THF, ether, 0 °C. (d) NH3, MeOH, THF, rt; (e) RB(OH)2,
Pd(PPh3)4, Na2CO3, dioxane, H2O, 80 °C.
B
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iodoisocyanates or iodoisothiocyanates. Immediate reaction
with ammonia provided the desired aminooxazoline21 and
aminothiazoline, respectively. A Suzuki reaction provided the
desired analogues 57, 58, and 61−63.
Synthesis of the six-membered amino heterocycles com-
menced from ketone 2c (Scheme 4). Vinyl Grignard addition,
the spiro-bicyclic core precludes direct trajectory into this
pocket from the 5, 7, and 8 positions (Figure 1). The 6
position, in contrast, is ideally positioned for extending into the
S3 pocket. To test this hypothesis, a range of 6-position
analogues were prepared, resulting in 4- to 3600-fold
improvements in BACE1 potency (Table 1).
The potency of unsubstituted phenyl analogue 11 (BACE1
IC50 = 455 nM) was further improved by meta substitution24
with lipophilic and hydrophilic groups (Table 1). For example,
the 3-chlorophenyl group improved BACE1 potency to 24 nM
and the more polar 3-cyano- and 3-methoxyphenyl improved
potency to 94 and 68 nM (compounds 12, 13, and 14,
respectively). In contrast, ortho and para substitutions (15 and
16) resulted in a significant loss of potency vs the unsubstituted
phenyl of 11. The preference for meta substitution is
understood by examining the X-ray BACE1 cocrystal structures
of 12 and 13 (Figure 2) wherein the m-Cl and m-CN groups
are readily accommodated in the BACE1 protein. In contrast,
little room is available for ortho and para substitution of the
phenyl ring. For similar reasons, additional phenyl substitution
(compounds 17−21) is not significantly beneficial.
Scheme 4. Synthesis of Aminothiazine and
Aminodihydropyrimidinone Analogues
a
Larger nonpolar meta substituents generally decreased
BACE1 affinity and/or increased the enzyme to cell shift. For
example, the 3-isopropoxy analogue 22 lost 40-fold BACE1
potency vs the 3-methoxy analogue 14, while the 3-ethoxy
analogue 23 lost significant cellular potency vs 14 (633 nM vs
164 nM, respectively). Similarly, the 3-CF3 and 3-SMe
analogues 24 and 25 were inferior to the 3-Cl analogue 12
(166 and 932 nM vs 107 nM, respectively). In contrast,
difluorination of the 3-methoxy group of analogue 14 provided
∼2-fold potency improvement for analogue 26. No benefit was
seen with the smaller 3-F analogue 27 vs 12; however, the 3-
CN analogue 13 provided improved cellular potency vs 12 (36
vs 107 nM). The BACE1 cocrystal structures of 12 and 13
again provide insight into these trends as the pocket accepting
the meta substituents is limited in width (decreased BACE1
potency for 22 and 25) but extends deeply enough to accept
the linear nitrile of 13.
Screening of nonaromatic P3 groups proved to be less
productive than for the aryls. The best results were found with
cyclohexyl 28 and ether 29. Efforts to improve the potency of
the cyclohexyl group by substitution (methylation of the 3 or
the 3,5 position; data not shown) were not productive, as there
is limited room in the S3 pocket (Figure 2). The intolerance for
the piperidine moiety of analogue 30 (8661 nM) is likely due to
the high lipophilicity of residues in the region near the
piperidine nitrogen.
a
Reagents and conditions: (a) vinylmagnesium bromide, THF, 0 °C;
(b) Thionyl chloride, hexanes, 0 °C; (c) thiourea, CH3CN, 0−50 °C;
(d) TFA, MsOH, 0 °C to rt; (e) 5-chloropyridine-3-boronic acid,
PdCl2(dppf)·DCM, Na2CO3, dioxane, H2O, 80 °C; (f) BuSONH2,
Ti(OEt)4, THF, reflux; (g) methyl acetate, LDA, THF, −78 °C; (h)
HCl, dioxane, rt; (i) EDCI, 10, Pr2EtN, DMF, rt.
t
i
thionyl chloride treatment, and thiourea displacement of the
allylic chloride provided carbamimidothioate 7. Cyclization to
the aminothiazine was accomplished at low temperature with a
1:1 mixture of methanesulfonic acid and TFA.22 A Suzuki
reaction provided thiazine 59. Ellman−Tang methodology
provided β-amino ester equivalent 8 from ketone 2c.23
Deprotection followed by cyclization with protected thiourea
10 and EDCI provided bromide 9. Standard Suzuki reaction
conditions constructed the biaryl bond and removed the Boc
protecting group to provide analogue 60.
All analogues were synthesized and assayed initially as
racemates. Analogues with a range of BACE1 potency,
selectivity vs CatD, permeability, efflux liability, and microsomal
stability were assessed for BBB permeability in mice.
Compounds achieving successful profiles including promising
unbound concentrations in the brain (Cfree,brain) were resolved
and advanced to pharmacodynamic studies.
For pyridine analogues, the 3-pyridyl isomer was substantially
(20- to 55-fold) more potent than the 2- and 4-pyridyl isomers
(Table 1, 31 vs 32 and 33). Examination of BACE1 cocrystal
structures (not shown) of nitrogen containing heterocycles
revealed a water bridging the nitrogen of pyridin-3-yl and
pyrimidin-5-yl analogues to Ser229. As previously described,
this through-water interaction explains, at least in part, the
preference for the 3-pyridyl isomer 31.24
Combining the 3-pyridyl water bridge to Ser229 with the
preference for meta substitution observed for analogue 12 led
to the discovery of cell potent (30−47 nM) analogues 34−36.
The 5-chloropyridin-3-yl 34 was particularly attractive, as it
maintained good enzyme potency (34 nM), low MW (370.8),
limited molecular flexibility, and reasonable polarity (tPSA =
80). Additionally, 34 did not exhibit time dependent inhibition
of cytochrome P450 enzymes, an advantage over alkyne 36.
RESULTS AND DISCUSSION
■
Primary assays for our BACE program included BACE1
potency, CatD selectivity, and efflux liability. BACE1 and
CatD potencies were based upon the cleavage of relevant
peptide substrates, while efflux liability was assessed in LLC-
PK1 cells transfected with human MDR1 gene. Prototype
compounds and analogues meeting a potency threshold of
<1000 nM progressed to a cell-based Aβ1−40 inhibition assay.
HEK-293 cells expressing wild type APP were treated with
compound, and production of Aβ1−40 was quantified with an
HTRF assay (Cisbio). Compound toxicity was assessed using
the CellTiter-Blue assay (Promega) as well by visual assessment
of morphological change.
The X-ray structure of 1a reveals an opportunity to increase
BACE1 potency by filling the S3 pocket, though the rigidity of
C
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a
Table 1. S3 Exploration with Spiro-aminohydantoin-chroman
a
b
c
Data for racemates. Mean IC50 of at least two independent experiments. Mean EC50 of at least two independent experiments for cellular Aβ
d
e
e
production. (B to A)/(A to B) value derived for compounds (1 μM) evaluated in LLC-PK1 cells transfected with human MDR1 (Pgp). Apical
(A) and basolateral (B). Not determined.
f
D
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electron rich core. Full details of that work will be reported in a
subsequent publication.
In an attempt to further reduce efflux and increase potency,
our SAR exploration was extended from the chroman to the
thiochroman and tetrahydronaphthalene (THN) cores. The
SAR for the chroman, thiochroman, and THN cores (Table 2)
was similar for BACE1 enzyme potency, CatD enzyme potency,
and efflux. The cellular potency was found to be slightly
divergent, with differences up to 6-fold favoring the THN core
when compared to the chroman core (46 vs 37, 48 vs 34, and
50 vs 39). The improved cellular potency of the THN core
brought compounds such as pyrimidine 50 into consideration
for in vivo studies. Attempts to further increase the potency of
the THN core by benzylic substitution (ketone 51 and difluoro
analogue 52) led to significant losses in potency and selectivity.
Although we were pleased overall with the low MW, limited
molecular flexibility, potency, and selectivity found in these
cores, the efflux liability remained. We expected that analogues
with tPSA of <70 would reach minimal efflux ratios of <2.26
Unfortunately, this effort did not reach fruition, as even
compounds with a tPSA value of <60 (analogues 42 and 43)
remained efflux substrates (efflux ratios of 4.8 and 5.7,
respectively). We hypothesized that the compact polarity of
the aminohydantoin (tPSA = 59) minimized the opportunity to
identify efflux free analogues.
As core manipulation was not successful for avoiding efflux,
we turned our attention to decreasing the polarity of the Asp
binding portion of the molecule to enable CNS exposure goals.
Our first approach was an attempt to shield the compact
polarity of the aminohydantoin with larger alkyl groups. As
Table 3 shows, this effort did in fact result in improved efflux
ratios of 2.8 and 1.8 for analogues 53 and 54, respectively.
Unfortunately, the improvement in efflux came with a
significant loss of BACE1 potency and selectivity vs CatD.
Further elaboration of the aminohydantoin to acylpiperidine 56
did rescue BACE1 potency; however, the compound equally
inhibited CatD and the efflux liability returned.
In parallel, the polarity of the Asp binding moiety was
directly modified by removing the amide carbonyl (Table 3,
analogues 57−59, 61−63). Of the analogues shown in Table 3,
only the thiazoline analogue 58 approached an efflux ratio of
<2. It was disappointing that once again an improved efflux
ratio came with greatly diminished BACE1 potency. Also, one
carbon homologation of the aminohydantoin 48 (Table 2) to
the aminopyrimidinone 60 (Table 3) resulted in a 1000-fold
loss of potency and no improvement in efflux, a significant
disconnect from the pioneering work of Astra Zeneca and
Astex17a as well as the optimization studies of Merck.27
Expansion of the oxazoline SAR to other P3 groups (Table 3,
analogues 61−63) did not result in improved BACE1 potency.
Examination of cocrystal structures of BACE1 with these
analogues did not reveal any loss of favorable interactions with
the protein or the introduction of any unfavorable interactions.
It is interesting to note that each of the oxazoline, thiazoline,
and thiazine analogues demonstrated a significant and favorable
potency shift (up to 26-fold) from the enzyme to cellular Aβ
production.28
Figure 2. X-ray cocrystal structures of BACE1 and 12, 13, and (R)-50.
Only density for the (R)-enantiomers of racemates 12 and 13 was
detectable in the BACE1 protein.
Combining the 3-pyridyl water bridge with either a 2- or 5-
fluoro was not as advantageous as introduction of the 5-Cl (37
and 38 vs 34).
The low MW (337.4) and good CatD selectivity (97-fold) of
the pyrimidine analogue 39 was appealing, despite the
moderate potency. Introduction of a 2- or 4-methyl led to
>5-fold loss in potency (data not shown) as predicted by the
limited room available to these positions in the S3 pocket.
However, the discovery that the pyrimidine moiety engages
Gly11 through a bound water (exemplified with (R)-50, Figure
2) and provides good CatD selectivity maintained our interest
in this P3 moiety as we explored other cores.
Seeking to capitalize on potential through-water interactions,
we expanded the search to five-membered heterocycles. The
most promising result from this effort was the isothiazole
analogue 40 (501 nM). Other regioisomers and substitutions
were not helpful, indicating that the nitrogen of the six-
membered heterocycles is ideally directed for the through-water
interaction.
As potency and efflux were primary concerns for efficacy,
selectivity vs CatD was necessary for tolerability.25 In general,
carbocyclic P3 groups provided compounds with poor
selectivity vs CatD (<1 to 31-fold), while six-membered
heterocycles showed greater promise (up to 97-fold). The
greatest selectivity (784-fold) was achieved with furanylamide
41. The observation that amide 41 possessed extremely weak
CatD activity (180 420 nM) led to a parallel effort on a less
Compounds of varying permeability, efflux liability, potency,
selectivity, and liver microsomal stability were evaluated in the
mouse to assess brain disposition (Table 4). Although close
attention was paid to bioavailability, clearance, and plasma
exposure, our primary focus was the free drug concentration in
the brain. More specifically, we were concerned with the ratio
E
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a
Table 2. Chroman, Thiochroman, and THN SAR
a
b
c
Data for racemates. Mean IC50 of at least two independent experiments. Mean EC50 of at least two independent experiments for cellular Aβ
d
production. (B to A)/(A to B) value derived for compounds (1 μM) evaluated in LLC-PK1 cells transfected with human MDR1 (Pgp).
of the free brain concentration to the cell IC50. This “coverage
ratio” was a key assessment that incorporated PK, potency, and
brain penetration. This ratio was calculated for the 1 and 4 h
time points and is found in the bottom line of Table 4. The
team’s goal was to identify a compound with free brain
exposure within range of its cellular potency when dosed orally
(10 mg/kg), a significant hurdle for an efflux substrate.
The data in Table 4 illustrate that despite efflux, conforma-
tionally constrained, permeable29 compounds can effectively
penetrate the BBB. Potency alone was not the most critical
variable, as the most potent compound (48) had the poorest
coverage ratio due to rapid CL, and the least potent compound
evaluated (63) was one of the best at the 1 h time point
(coverage ratio of 0.38). Pyrimidine 50 achieved the best
overall coverage ratios (0.37 and 0.40) despite greater than liver
blood flow CL.30 Incorporating nonspecific binding improved
the correlation, as the compounds with the best free brain
concentration (34, 50, and 63) showed the lowest unbound CL
(CLu) and compounds (42, 48) with the highest CLu
demonstrated low free brain concentrations. From the
experimental results in Table 4, molecules with the best
balance of potency and in vivo stability (CLu) provided the best
coverage ratios.
were roughly twice as potent as the corresponding racemates
(Table 5). Fortunately, the (R)-enantiomers did not con-
sistently show increased affinity for CatD, effectively increasing
the CatD/BACE1 window for most compounds. The increase
in BACE1 affinity translated into improved cell potency for
each (R)-enantiomer evaluated raising promise for in vivo CNS
Aβ lowering. Liver microsomal assays predicted low to medium
CL across species (mouse, rat, guinea pig, cynomolgus monkey,
and human) for the compounds in Table 5 with the exception
of (R)-46 in the rat. Though pleased with predicted stability of
the single enantiomers, as well as the potency and selectivity
increases, all compounds remained efflux substrates. Pro-
gression to pharmacodynamic assays would ascertain if
permeable, low MW, limited molecular flexibility compounds
could affect CNS Aβ1−40 production despite the efflux liability.
The single enantiomers were evaluated for their ability to
achieve adequate Cfree,brain to lower CNS Aβ1−40 levels following
oral dosing, the key proof of mechanism data for progressing
lead compounds. For the majority of studies, the drug
concentration was determined for the plasma, CSF, and brain
compartments. The pharmacodynamic response (n = 6−8
animals per dose and time point) was measured as the
percentage of CSF Aβ1−40 vs the vehicle control (six to eight
animals). For studies over 5 h, two vehicle control groups were
utilized and assessed at the first and last time points.
The compounds in Table 4 demonstrating the best brain PK
were scaled, resolved, and profiled. We found that the BACE1
potency resided primarily within the (R)-enantiomers, as they
F
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a
Table 3. Asp Binding Elements: Potency and Efflux SAR
a
b
c
Data for racemates. Mean IC50 of at least two independent experiments. Mean EC50 of at least two independent experiments for cellular Aβ
d
e
production. (B to A)/(A to B) value derived for compounds (1 μM) evaluated in LLC-PK1 cells transfected with human MDR1 (Pgp). Not
determined.
The single enantiomers were progressed to single oral dose
pharmacodynamic assessment in the guinea pig, a preferred
rodent species due to the high homology with human APP.31
Utilizing wild type animals precluded the cost of transgenic
animals and the caveats associated with such strains.32 We
began with a dose response study of (R)-34 (Table 6).33
Chloropyridine analogue (R)-34 decreased CSF Aβ1−40 levels
at all doses from 25 to 400 mg/kg. Reduction of CSF Aβ1−40 by
50% correlated with Cfree,brain that exceeded 2-fold the
compound’s cellular IC50 (coverage ratio of >2). Increasing
the dose to 200 mg/kg resulted in a coverage ratio of 16 and
CSF Aβ1−40 reduction of 71%. Interestingly, increasing the dose
to 400 mg/kg did not result in further CSF Aβ1−40 reduction
despite doubling the coverage ratio to 33. It is possible that
other secretases34 are responsible for the remaining Aβ1−40
production in the guinea pig.
The next compound evaluated, the benzonitrile (R)-45,
reduced CSF Aβ1−40 by only 29% with a dose of 150 mg/kg.
The F-pyridine analogue (R)-46 demonstrated 50% CSF
Aβ1−40 reduction at 150 mg/kg but no effect at 75 mg/kg
where the coverage ratio was only 1.2. The inferior results with
these two compounds precluded further studies, including full
evaluation of brain disposition for (R)-45.
(R)-50 was evaluated in a time course study. We were pleased
to find that 60 mg/kg (R)-50 was sufficient to reduce CSF
Aβ1−40 by 50% at the 5 h time point with a coverage ratio of 4.4.
Significant reduction (20−43%) was also observed at the other
time points, including 12 h after dose (23% reduction with a
coverage ratio of 1.5).
Moving from the aminohydantoin to the aminooxazoline
heterocycles, (R)-63 was evaluated in the guinea pig at 3 h with
doses of 50 and 150 mg/kg. Significant CSF Aβ1−40 reduction
was observed with both doses, although the 50 mg/kg dose
provided only a modest CSF Aβ1−40 reduction of 30%. Also,
(R)-63 was poorly soluble and required a less preferred
formulation (PEG400/ethanol/water), decreasing enthusiasm
for additional work with this analogue, including determining
brain concentrations and coverage ratios.
The free brain-to-free plasma ratios for all compounds
evaluated from this series failed to reach unity in the guinea pig.
While the poor free brain-to-free plasma ratios were predicted
by the efflux data and the mouse PK results, we were surprised
at the difference between the CCSF and Cfree,brain (Table 6).
Although the CSF is often utilized as a surrogate for free brain
levels,35 our observations suggest a clear difference in the
guinea pig blood−CSF barrier vs the BBB. A similar disconnect
has been reported in the mouse for efflux substrates.36
In parallel, we evaluated (R)-34 and (R)-50 in a rat
pharmacodynamic model.37 Both compounds were assayed in a
time course study to correlate Cfree,brain to CSF Aβ1−40 response
(Table 7).38 At 150 mg/kg, (R)-34 reduced CSF Aβ1−40 levels
at a maximum of 29% at 3 h. The Cfree,brain at this point was >50
Early results with the pyrimidine analogue (R)-50 were
encouraging, resulting in both time course and dose response
studies. In the dose response study, an oral dose of 100 mg/kg
resulted in 55% CSF Aβ1−40 reduction at 3 h, a decreased effect
(23%) with 30 mg/kg, and no effect with the 10 mg/kg dose.
With these promising data, an intermediate dose of 60 mg/kg
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a
b
Table 4. In Vitro Properties and Brain and Plasma PK (3 mg/kg iv and 10 mg/kg po)
a
b
1
Compounds were racemic and >95% pure by H NMR and HPLC. iv formulation: 40:10:50 PEG400/ethanol/H2O. po formulation: 1%
c
carboxymethylcellulose (CMC), 0.5% polysorbate 80 (Tween 80), 5 mM citrate, pH 4, in water. Mean EC50 of at least two independent
experiments for cellular Aβ production. A to B in parental LLC-PK1 cell line. Efflux ratio is the (B to A)/(A to B) value derived from LLC-PK1
cells transfected with human MDR1 (Pgp). Compounds (1 μM) incubated with mouse liver microsomes for 20 min at 37 °C in the presence of
NADPH. CLu = CL/fu,plasma
d
e
f
g
h
i
j
. po dose. Cfree,brain/(cell IC50). Below level of quantification.
nM (coverage ratio of 3.8), which was greater than the
concentration (32 nM) that provided 51% CSF Aβ1−40
reduction in the guinea pig, an intriguing disconnect.
Pyrimidine (R)-50 provided similar reduction to (R)-34 but
with a smaller dose (60 mg/kg). The maximal effect of 24%
CSF Aβ1−40 reduction was achieved at 5 h with Cfree,brain of 50
nM (R)-50. This compared well with the guinea pig result of
from baseline, and the drug CCSF was plotted from 0 to 48 h
(Figure 3). A 100 mg/kg oral dose of (R)-50 achieved >65%
CSF Aβ1−40 reduction at 8 h, approximately 4 h after Cmax. The
duration of response was quite robust, as the CSF Aβ1−40 levels
in the dose group had not returned to predose levels by 48 h.
The results were encouraging, though the variability in the
measurement of CSF Aβ1−40 was a significant challenge. Full
experimental details of this study will be discussed elsewhere.
23% CSF Aβ1−40 reduction for the 30 mg/kg dose (Cfree,brain
=
53 nM).
As with the guinea pig, the variance in drug exposure in the
CSF vs the brain suggests a difference in the rat blood−CSF
barrier and BBB with these analogues. This disconnect greatly
limits the ability to predict doses in higher species, particularly
in humans. Eliminating efflux would address this liability,
increase brain exposure, and decrease the likelihood of
peripheral adverse events.39
Despite the efflux and moderate CSF Aβ1−40 reduction in the
rat, we were pleased to demonstrate proof of mechanism in two
separate rodent species. These data, coupled with a >100-fold
BACE1 vs CatD selectivity, led us to study (R)-50 in a higher
species. As a previous report utilized the monkey to measure
Aβ lowering with a BACE1 inhibitor (co-dosed with a CYP
inhibitor),40 we utilized this species for further pharmacological
assessment.
CONCLUSION
■
We describe the utilization of structure-based design,
conformation constraint, and wild type animal models to
identify potent, selective, and brain penetrable inhibitors of the
BACE1 enzyme. Though unable to identify potent alternative
Asp binding moieties to eliminate efflux, we discovered
analogues that decreased CSF Aβ1−40 in nontransgenic rodent
species. The CSF Aβ1−40 response was directly related to free
drug concentrations in the brain in the rat and guinea pig.
Further proof of mechanism was demonstrated in the
cynomolgus monkey with analogue (R)-50, providing >65%
CSF Aβ1−40 reduction at the 8 h time point. Efforts to optimize
BACE1 potency by utilizing P2′ interactions, increase CatD
selectivity with non-aniline amides, and eliminate efflux will be
published in due course.
The pharmacodynamic response (CSF Aβ1−40 lowering) in
the cynomolgus monkey was measured as a percentage change
H
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Table 5. Single Enantiomer Profiles
a
b
c
Mean IC50 of at least two independent experiments. Mean EC50 of at least two independent experiments for cellular Aβ production. A to B in
d
parental LLC-PK1 cell line. Efflux ratio is the (B to A)/(A to B) value derived from LLC-PK1 cells transfected with human MDR1 (Pgp).
e
Compounds (1 μM) incubated for 20 min at 37 °C in the presence of NADPH with mouse (M), rat (R), guinea pig (GP), cynomolgus monkey
(C), or human (H) liver microsomes (LM).
Table 6. Guinea Pig Pharmacodynamics (Aβ1−40 Lowering)
dose (po)
(mg/kg)
time point
(h)
Cfree,plasma
(nM)
Cfree,brain
(nM)
free
(b/p)
CCSF
(nM)
coverage
b
CSF Aβ
1−40 c
a
compd
formulation
ratio
reduction (%)
(R)-34
1% CMC/0.5% Tween 80
25
5
5
5
5
5
5
5
5
3
3
3
1
3
5
8
12
3
33
6.3
0.19
10
0.50
2.3
7.5
16
31
51
53
71
72
29
0
50
224
1209
2891
4962
122
304
562
15
32
0.14
86
100
200
400
150
75
105
224
456
0.087
0.077
0.091
456
1321
2384
195
20
33
e
(R)-45
(R)-46
1% CMC/0.5% Tween 80
ND
d
0.5% HPMC /0.5% Tween 80
11
0.037
1.2
e
150
10
ND
247
27
50
0
(R)-50
1% CMC/0.5% Tween 80
22
0.68
0.81
2.0
8.5
5.2
6.3
4.4
1.7
1.5
30
74
53
0.72
125
1620
2300
920
440
66
23
55
20
43
50
35
23
30
100
60
913
5380
1940
534
127
66
230
146
176
124
48
0.25
0.027
0.091
0.232
0.378
0.622
60
60
60
60
41
47
e
(R)-63
40/10/50 PEG400/ethanol/H2O
50
3019
6673
ND
888
5609
e
150
3
ND
70
a
b
c
d
e
1
Purity of >95% by H NMR and HPLC. Cfree,brain/(cell IC50). Reduction vs vehicle control. Hydroxypropylmethylcellulose (HPMC). Not
determined.
protein was concentrated to 10 mg/mL and frozen for crystallographic
studies.
EXPERIMENTAL SECTION
■
X-ray Crystal Structure. Human BACE1 (residues 57−453) was
Crystals were grown by hanging drop vapor diffusion with a
precipitant of 20% PEG200, 0.1 M sodium acetate, pH 4.5−5.6, and
0−2 mM cobalt chloride. Crystals grew in space group C2221 in
approximately 1 week. Crystals were then transferred to a soaking
solution (27−30% PEG200, 0.1 M NaOAc, pH 5.0−5.6, 0−2 mM
CoCl2, and 10% DMSO) with compound at a final concentration of 1
mM. Crystals were soaked for 18−24 h and either flash-frozen in liquid
nitrogen or mounted directly for data collection. The soaking solution
also served as a cryprotectant for data collection at 100 K.
expressed in E. coli with a noncleavable 6-His tag at the C-terminus.
The protein was refolded and purified in a manner similar to that from
Tomasselli et al.41 Briefly, cells were lysed using mechanical disruption
and inclusion bodies recovered by differential centrifugation. Inclusion
body proteins were solubilized in urea and refolded by dilution into
water. Following a 3 week refold period, BACE protein was recovered
by Q-Sepharose anion-exchange chromatography. Fractions containing
active protein were pooled and further purified over Source-Q anion-
exchange and Sephadex 200 size-exclusion columns. Purified BACE
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Table 7. Rat Pharmacodynamics (Aβ1‑40 Lowering)
dose (po)
(mg/kg)
time point
(h)
Cfree,plasma
(nM)
Cfree,brain
(nM)
free
(b/p)
CCSF
(nM)
coverage
b
CSF Aβ
reduction
1−40 c
a
compd
formulation
ratio
(%)
(R)-34
1% CMC/0.5% Tween 80
150
1
1166
1023
1254
1353
2442
1733
561
42
53
56
68
78
90
50
39
26
0.036
0.052
0.044
0.050
0.032
0.052
0.089
0.67
8330
15600
24200
2270
269
3.0
3.8
4.0
4.9
2.9
3.3
1.9
1.4
1.0
12
29
20
18
16
17
24
3
5
8
(R)-50
1% CMC/0.5% Tween 80
60
1
3
236
5
47
d
8
57
15
ND
13
12
24
1.09
11
a
b
c
d
1
Purity of >95% by H NMR and HPLC. Cfree,brain/(cell IC50). Reduction vs vehicle control. Not determined.
calculated from the fluorescence increase. The final concentrations
were 10 nM enzyme and 2 μM substrate.
Cell-Based Assay. Human embryonic kidney cells (HEK293)
stably expressing APPwt were plated at a density of 35K cells/well in
96-well collagen coated plates (BD-Biosciences). The cells were
cultivated overnight at 37 °C and 5% CO2 in DMEM supplemented
with 10% FBS. The following day the medium was changed and the
cells were incubated for 48 h with test compounds at concentrations
ranging from 0.0008 to 16 μM. Following incubation with the test
compounds the conditioned medium was collected and the Aβ1−40
levels were determined using an HTRF assay (CisBio). The IC50 was
calculated from the percent of control Aβ40 as a function of the
concentration of the test compound. The HTRF to detect Aβ1−40 was
performed in 384-well microtiter plates (Corning). The antibody pair
that was used to detect Aβ1−40 from cell supernatants was a pair of
monoclonal antibodies, one labeled with cryptate and one labeled with
XL655 as supplied by the manufacturer (CisBio). Conditioned
medium was incubated with antibody pair overnight at 4 °C. The
plates were measured for time-resolved fluorescence on a Victor2 plate
reader (Wallac).
Figure 3. Monkey pharmacodynamics of (R)-50 (Aβ1−40 lowering).
CSF concentration of Aβ1−40 (baseline normalized, left Y-axis, broken
line) and (R)-50 (right Y-axis, solid line) following single 100 mg/kg
oral dose of (R)-50 in cynomolgus monkeys. Data points represent
mean and SD from 12 animals.
Permeability and Efflux Assay. The LLC-PK1 cell line, stably
transfected with human MDR1 cDNA, was obtained from BD
Biosciences (Franklin Lakes, NJ). Both parental LLC-PK1 and MDR1
transfected LLC-PK1 cells were cultured and plated according to the
manufacturer’s recommendations with the exception that the passage
medium contained only 2% fetal bovine serum to extend passage time
out to 7 days. The cells were plated into Millicell-96 cell culture insert
plates at a density of 50 000 and 40 000 cells/well for the MDR1 and
parental LLC-PK1 cell lines, respectively. The plates were incubated at
37 °C and in an atmosphere of 5% CO2 with saturating humidity. The
medium was replaced on day 2 of culture. Permeability experiments
were conducted on culture day 6. A trans-epithelial electrical resistance
(TEER) value of at least 500 Ω as determined by the Millipore
Millicell-ERS TEER device (Millipore, Billerica, MA) was required for
monolayer acceptance. Transport buffer was prepared using Hanks
balanced salt solution (HBSS), 1% DMSO, and 10 mM HEPES. The
pH of the assay buffer was adjusted to 7.4, using 1.0 M KOH. Stock
solutions for the test compounds were prepared in DMSO for final
concentration of 1 μM. Final organic concentration in the assay was
1%. Bidirectional permeability was assessed by adding either 75 or 250
μL of buffer containing compound to the apical or basolateral
compartments, respectively. Buffer only was added to the receiver
compartment. All tests were performed in triplicate, and compounds
were assayed for both apical to basolateral (A to B) and basolateral to
apical (B to A) transport. After a 2 h incubation on an orbital plate
shaker (VWR, West Chester, PA) at 50 rpm and 37 °C with 5% CO2,
the culture plates were removed and an amount of 50 μL was sampled
from the apical and basolateral portion of each well and added to a
plate containing 150 μL of 1 μM labetalol (internal standard) in 2:1
acetonitrile (ACN)/H2O, v/v. The plates were read using a Molecular
Devices (Sunnyvale, CA) Gemini fluorometer to evaluate the lucifer
yellow concentrations at excitation/emission wavelengths of 425/535
nm. These values were accepted when found to be below 5% for apical
to basolateral and basolateral to apical flux across the MDR1
Diffraction data were collected on a Rigaku FR-E generator and R-
axis IV++ detector (Rigaku Inc., 9009 New Trails Drive, The
Woodlands, TX 77381-5209, U.S.). Data were processed in Moslfm,42
and the initial structure was solved by molecular replacement from
1FNK.pdb using Molrep. All crystal structures were refined in Refmac,
all three of these programs being part of the CCP4 suite (Winn et al.,
2011).43 Model building was performed in Coot.44
BACE1 Enzymatic Assay. The activity of purified recombinant
human BACE1, expressed in CHO cells, was determined using a
custom synthesized biotinylated peptide based upon sAPP. Uncleaved
substrate was detected via HTRF whereby europium-labeled anti-Aβ
(amino acids 1−17, clone 6E10) was combined with D2-labeled
streptavidin. Specifically, 20 nM enzyme was incubated with 150 nM
peptide (biotin-Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn-Leu-Asp-Ala-
Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu, rep-
resenting P10−P17′) for 6 h at 22 °C in an assay buffer consisting of
50 mM sodium acetate (pH 4.4) and 0.1% CHAPS. In IC50 studies,
compounds were serially diluted (1:2.5) in DMSO (11 points),
compound and enzyme were preincubated for 15 min at room
temperature, and the enzymatic reaction was initiated with peptide.
The detection solution consisted of 43 nM streptavidin-D2 and 1.1
nM antibody in 200 mM Tris (pH 8.0), 20 mM EDTA (pH 8.0), 0.1%
BSA, and 0.8 M KF. Following incubation for 2 h at 22 °C, the
samples were excited at 320 nm and the emission ratio of 665 nm/615
nm was determined.
CatD Enzymatic Assay. The catalytic activity of cathepsin D,
purified from human spleen (Calbiochem), was assessed in a FRET
peptide substrate assay using 50 mM sodium acetate (pH 4.4) and
0.1% CHAPS. In IC50 studies, compounds were serially diluted (1:2.5)
in DMSO (11 points). The substrate (Anaspec catalog no. 72097)
contains a 5-FAM/QXL 520 pair whereby cleavage results in an
increase of 5-FAM fluorescence that is measured using an excitation of
485 nm and an emission of 535 nm. The fluorescence was monitored
continuously for 1 h at room temperature, and initial rates were
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transfected LLC-PK1 cell monolayers. The compound concentrations
were determined from the ratio of the peak areas of the compound to
labetalol in comparison to the dosing solution. The LC−MS/MS
system comprised an HTS-PAL autosampler (Leap Technologies,
Carrboro, NC), an HP1200 HPLC (Agilent, Palo Alto, CA), and a
MDS Sciex 4000 Q trap system (Applied Biosystems, Foster City,
CA). Mass spectrometric detection of the analytes was accomplished
using the ion spray positive mode. Analyte responses were measured
by multiple reaction monitoring (MRM) of transitions unique to each
compound in comparison to labetalol. Permeability (Papp) was
calculated in BioAssay, version 9.0 (Cambridge Soft, Cambridge,
MA) using the following equation:
concentration in rat and guinea pig CSF samples was analyzed by
MSD 96-well MULTI-ARRAY human/rodent (4G8) Aβ 40 ultra-
sensitive assay (rat) or human (6E10) Aβ 40 ultrasensitive kit (Meso
Scale Discovery, Gaithersburg, MD). The 96-well MULTI-SPOT
Aβ1−40 peptide plate was incubated with 1% BSA solution at room
temperature with shaking for 1 h and washed per manufacturer’s
instructions with Tris wash buffer in deionized water. The detection
antibody solution (25 μL/well, sulfo-tag 4G8 in 1% BSA) and CSF
samples or standards (25 μL/well) were added to the plate for
incubation at room temperature for 2 h. The plate was washed three
times before addition of MSD read buffer and was read on Sector
Imager 2400 (Meso Scale Discovery, Gaithersburg, MD) immediately
afterward. The Aβ1−40 levels obtained as pg/mL were analyzed by two-
tailed Student’s t test to compare mean percentage Aβ1−40 reduction in
vehicle-treated control animals and compound-treated animals.
Chemistry. Unless otherwise noted, all materials were obtained
from commercial suppliers and used without further purification.
Anhydrous solvents were obtained from EM Science and used directly.
All reactions involving air- or moisture-sensitive reagents were
performed under a nitrogen or argon atmosphere. All final compounds
were purified to ≥95% purity as determined by high-performance
liquid chromatography (HPLC). Purity was measured using either
system A (Varian ProStar with UV detection at 220 and 254 nm, a
Waters YMC ODS-AQ C18 3.0 mm × 50 mm, 3.0 μm, 5−95%
CH3CN in H2O with 0.1% NH4OAc for 5 min at 1.0 mL/min) or
system B (Varian ProStar with UV detection at 220 and 254 nm, a
Waters YMC ODS-AQ C18 3.0 mm × 50 mm, 3.0 μm, 5−95%
CH3CN in H2O with 0.1% formic acid for 5 min at 1.0 mL/min).
Silica gel columns were used with prepacked silica gel cartridges
CdV(1 × 106)
P
(×10−6 cm/s) =
app
(t)(0.12 cm2)(C0)
where Cd, V, t, and C0 are the detected concentration (μM), the
volume on the dosing side (mL), the incubation time (s), and the
initial dosing concentration (μM), respectively. The calculations for
Papp were made for each replicate and then averaged. The efflux ratio
(ER) was calculated from the basolateral to apical transport divided by
the apical to basolateral transport:
P (B→A)
app
ER =
P (A→B)
app
Microsomal Stability Assay. Compounds were prepared in a 100
mM potassium phosphate buffer solution (pH 7.4) for a final
concentration of 1 μM and incubated in the presence of 1 mg/mL
human, monkey, rat, guinea pig, or mouse liver microsomes. The final
concentration of DMSO in the incubation was less than 0.1%.
Metabolism was initiated with the addition of a 10 μL aliquot of
NADPH-regenerating solution (20 mM MgCl2 buffer containing
NADP (17 mg/mL stock in 100 mM KH2PO4), glucose 6-phosphate
(78 mg/mL stock in 100 mM KH2PO4), and glucose 6-phosphate
dehydrogenase (150 U/mL stock in 100 mM KH2PO4)) for a total
incubation volume of 50 μL. After 20 min of incubation at 37 °C and
100% relative humidity, metabolism was stopped with the addition of
150 μL of 60% acetonitrile containing internal standard (0.25 μM
labetalol). The plate was immediately spun in a centrifuge at 2095g for
7 min at room temperature. A 200 μL aliquot of the supernatant was
transferred from each well to a 96-well shallow plate, sealed, and
analyzed by LC−MS/MS.
Pharmacodynamic Assays. Male Sprague−Dawley rats and male
Hartley guinea pigs (6−8 weeks old, 250−350 g) were purchased from
Harlan (Indianapolis, IN) and Charles River Laboratories (Kingston,
NY), respectively. The animals were housed in groups of 3 at
controlled temperature and humidity in an alternating 12 h light and
dark cycle with free access to food and water in accordance with Array
BioPharma Institutional Animal Care and Use Committee Guidelines
and in harmony with the Guide for Laboratory Animal Care and Use.
Rats and guinea pigs (6−8 per group) received orally either vehicle or
compound at appropriate dose at a volume of 4−5 mL/kg. The rats
were euthanized by CO2 inhalation, and guinea pigs were euthanized
by euthasol (250 mg/kg) via intraperitoneal injection at predeter-
mined time points after dosing. The whole blood samples were
collected post-mortem via cardiac puncture in tubes containing 1.5%
EDTA, centrifuged at 14 000 rpm for 10 min at 4 °C. The plasma was
stored at −20 °C until drug concentration analysis. The CSF samples
(40−100 μL) were drawn post-mortem via cisterna magna puncture
with a 25 gauge needle (Becton Dickinson, Franklin Lakes, NJ) into
tubes containing 50 μL of 10% BSA in water. The CSF samples were
snap frozen in liquid nitrogen and stored at −80 °C until Aβ1−40
analysis. Sample weights were recorded to determine the actual
volume of CSF collected. The post-mortem animals were perfused
with 20 mL of sterile saline. Brain hemispheres were dissected (brain
stem and cerebellum were not collected), halved longitudinally, and
weighed, and only the left half of the brain was placed in a 15 mL
conical tube and snap frozen in liquid nitrogen. All tissues were stored
at −80 °C until drug concentration determination. CSF Aβ1−40
1
(Biotage or Teledyne Isco). H NMR spectra were recorded on a
Varian Mercury 400 (400 MHz) spectrometer or Varian Inova 400
(500 MHz) spectrometer at 25 °C. NMR spectra were processed using
VNMRj, version 2.1b. All observed protons are reported as parts per
million (ppm) downfield from tetramethylsilane (TMS) or other
internal reference in the appropriate solvent indicated. Data are
reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling
constants, and number of protons. Low-resolution mass spectrometry
(MS) data were determined on an Agilent 1100 series LCMS with UV
detection at 220 and 254 nm, a Halo C18 2.1 mm × 30 mm, 2.7 μm,
5−95% CH3CN in H2O with 0.1% NH4OAc for 3 min at 1.0 mL/min,
and atmospheric-pressure chemical ionization (APCI).
2′-Amino-6-bromo-1′,2,2-trimethylspiro[chroman-4,4′-imi-
dazol]-5′(1′H)-one (rac-1a). Step A. 6-Bromo-2,2-dimethylchro-
man-4-one (18.2 g, 71.3 mmol), KCN (9.29 g, 143 mmol), and
ammonium carbonate (48.0 g, 499 mmol) were diluted with
formamide (200 mL). The mixture was bubbled with argon for 15
min, sealed, heated to 70 °C, and stirred for 24 h. The mixture was
then heated to 110 °C and stirred for 48 h. The mixture was allowed
to cool and poured into ice−water (1L). The pH was adjusted to 5
with concentrated HCl, and the mixture was stirred for 1 h. The
precipitate was filtered and rinsed with water. The solid was air-dried
for 5 h and placed under vacuum for 15 h to afford 6-bromo-2,2-
dimethylspiro[chroman-4,4′-imidazolidine]-2′,5′-dione (21 g, 64.6
1
mmol, 90.5% yield) as a tan solid. H NMR (400 MHz, CDCl3-d) δ
7.34 (dd, J1 = 9.4 Hz, J2 = 2.3 Hz, 1H), 7.21 (d, J = 2.3 Hz, 1H), 6.76
(d, J = 9.4 Hz, 1H), 6.35 (br s, 1H), 2.6 (d, J = 14.9, 1H), 2.1 (d, J =
14.9 Hz, 1H), 1.51 (s, 3H), 1.31 (s, 3H); LCMS (APCI−) m/z 323,
325 (M − 1).
Step B. To a suspension of NaH (246 mg, 6.15 mmol) in DMF (6
mL) was added dropwise a solution of 6-bromo-2,2-dimethylspiro-
[chroman-4,4′-imidazolidine]-2′,5′-dione (1.0 g, 3.08 mmol) in DMF
(8 mL). After the mixture was stirred for 1 h, CH3I (0.198 mL, 3.08
mmol) was added, and the mixture was stirred for 16 h. The mixture
was diluted with DCM and washed with 1 N HCl, water, brine, dried
over MgSO4, filtered, and concentrated in vacuo. The isolated residue
was purified on silica gel, eluting with 5−100% ethyl acetate/hexanes
to yield 6-bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazolidine]-
1
2′,5′-dione (1.03 g, 3.04 mmol, 98.7% yield). H NMR (400 MHz,
CDCl3-d) δ 7.34 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.04 (d, J = 2.3 Hz,
K
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Journal of Medicinal Chemistry
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1H), 6.76 (d, J = 8.6 Hz, 1H), 6.1 (br s, 1H), 3.1 (s, 3H), 2.6 (d, J =
14.8, 1H), 2.1 (d, J = 14.8 Hz, 1H), 1.51 (s, 3H), 1.31 (s, 3H).
Step C. 6-Bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazoli-
dine]-2′,5′-dione (3.0 g, 8.8 mmol) and Lawesson’s reagent (2.1 g,
5.3 mmol) were diluted with toluene (50 mL) and heated under reflux
with a condenser and drying tube for 12 h. The mixture was allowed to
cool, diluted with ethyl acetate, and washed with 10% aqueous sodium
carbonate, water, and brine. The organic layer was separated, dried
(MgSO4), filtered, and concentrated. The residue obtained was
purified on silica gel, eluting with 5−50% ethyl acetate/hexanes to
yield 6-bromo-1′,2,2-trimethyl-2′-thioxospiro[chroman-4,4′-imidazoli-
din]-5′-one (2.0 g, 5.6 mmol, 64% yield). 1H NMR (400 MHz,
CDCl3-d) δ 7.35 (dd, J1 = 9.0 Hz, J2 = 2.3 Hz, 1H), 7.34 (br s, 1H),
6.94 (d, J = 2.3 Hz, 1H), 6.76 (d, J = 9.0 Hz, 1H), 3.34 (s, 3H), 2.56
(d, J = 14.8, 1H), 2.1 (d, J = 14.8 Hz, 1H), 1.51 (s, 3H), 1.35 (s, 3H).
Step D. A solution of 6-bromo-1′,2,2-trimethyl-2′-thioxospiro-
[chroman-4,4′-imidazolidin]-5′-one (2.0 g, 5.63 mmol) in methanol
(40 mL) was treated with tert-butyl hydroperoxide (11.7 mL, 84.4
mmol) followed by concentrated NH4OH (19.7 mL, 563 mmol). The
mixture was heated to 40 °C and stirred for 2 h. The mixture was
removed from the heat and stirred for an additional 2 h. The mixture
was diluted with water and extracted with DCM. The combined
organics were dried (MgSO4), filtered, and concentrated in vacuo. The
residue obtained was purified on silica gel, eluting with 1−10%
methanol/DCM with 1% NH4OH to yield 2′-amino-6-bromo-1′,2,2-
trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (1.5 g, 4.44
amino-6-(3-methoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imi-
dazol]-5′(1′H)-one (60 mg, 0.164 mmol, 25.9% yield). 1H NMR (400
MHz, CDCl3-d) δ 7.40 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.30 (t, J =
7.8 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 6.98 (m, 2H), 6.91 (d, J = 8.6
Hz, 1H), 6.84 (dd, J1 = 8.2 Hz, J2 = 2.7 Hz, 1H), 3.84 (s, 3H), 3.17 (s,
3H), 2.57 (d, J = 14.1 Hz, 1H), 1.99 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H),
1.40 (s, 3H); LCMS (APCI+) m/z 366.2 (M + 1).
2′-Amino-6-(2-methoxyphenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-15). rac-15 was synthe-
sized in the same fashion as compound 11, substituting 2-
methoxyphenylboronic acid for phenylboronic acid to afford 2′-
amino-6-(2-methoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imi-
1
dazol]-5′(1′H)-one (10 mg, 0.027 mmol, 19% yield). H NMR (400
MHz, CDCl3-d) δ 7.38 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.27 (m,
1H), 7.22 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.03 (br d, J = 1.9 Hz,
1H), 6.98 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 3.76 (s, 3H),
3.17 (s, 3H), 2.6 (d, J = 14.1 Hz, 1H), 1.99 (d, J = 14.1 Hz, 1H), 1.52
(s, 3H), 1.40 (s, 3H); LCMS (APCI+) m/z 366.2 (M + 1).
2′-Amino-6-(4-methoxyphenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-16). rac-16 was synthe-
sized in the same fashion as compound 11, substituting 4-
methoxyphenylboronic acid for phenylboronic acid to afford 2′-
amino-6-(4-methoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imi-
1
dazol]-5′(1′H)-one (18 mg, 0.049 mmol, 33% yield). H NMR (400
MHz, CDCl3-d) δ 7.35 (m, 3H), 6.91 (m, 4H), 3.82 (s, 3H), 3.19 (s,
3H), 2.57 (d, J = 14.1 Hz, 1H), 1.99 (d, J = 14.1 Hz, 1H), 1.51 (s, 3H),
1.40 (s, 3H); LCMS (APCI+) m/z 366.2 (M + 1).
1
mmol, 78.8% yield). H NMR (400 MHz, CD3OD-d4) δ 7.27 (d, J
= 8.6 Hz, 1H), 6.88 (s, 1H), 6.74 (d, J = 8.6 Hz, 1H), 3.14 (s, 3H),
2.31 (d, J = 14.1 Hz, 1H), 1.93 (d, J = 14.1 Hz, 1H), 1.45 (s, 3H), 1.36
(s, 3H); LCMS (APCI+) m/z 338.1 (M + 1).
2′-Amino-6-(3,5-dichlorophenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-17). rac-17 was synthe-
sized in the same fashion as compound 11, substituting 3,5-
dichlorophenylboronic acid for phenylboronic acid to afford 2′-
amino-6-(3,5-dichlorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imi-
(2′-Amino-6-(5-chloropyridin-3-yl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-11). 2′-Amino-6-
bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (50
mg, 0.15 mmol) (from step D of example 1a), phenylboronic acid (28
mg, 0.18 mmol), and tetrakis(triphenylphosphine)palladium(0) (8.5
mg, 0.0074 mmol) were combined in a vial and diluted with dioxane
(1 mL). Then 2 M aqueous Na2CO3 (370 μL, 0.74 mmol) was added,
and the vial was sealed and the mixture heated to 95 °C and stirred
overnight. The mixture was allowed to cool and loaded onto silica gel,
eluting with 1−10% MeOH/DCM with 1% NH4OH to yield 2′-
amino-6-(5-chloropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-
1
dazol]-5′(1′H)-one (20 mg, 0.05 mmol, 32% yield). H NMR (400
MHz, CDCl3-d) δ 7.34 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.29 (d, J =
1.9 Hz, 2H), 7.27 (d, J = 1.9 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.91
(br s, 1H), 3.20 (s, 3H), 2.54 (d, J = 14.1 Hz, 1H), 1.99 (d, J = 14.1
Hz, 1H), 1.52 (s, 3H), 1.42 (s, 3H); LCMS (APCI+) m/z 404.1 (M +
1)
2′-Amino-6-(3-fluoro-5-methoxyphenyl)-1′,2,2-
trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-18).
rac-18 was synthesized in the same fashion as compound 11, replacing
phenylboronic acid with (3-fluoro-5-methoxyphenyl)boronic acid to
afford 2′-amino-6-(3-fluoro-5-methoxyphenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (19 mg, 0.050 mmol, 34%
1
imidazol]-5′(1′H)-one (25 mg, 0.067 mmol, 46% yield). H NMR
(400 MHz, CDCl3-d) δ 7.40 (m, 5H), 7.30 (d, J = 8.6 Hz, 1H), 6.97
(s, 1H), 6.92 (d, J = 8.6 Hz, 1H), 3.18 (s, 3H), 2.55 (d, J = 14.1 Hz,
1H), 1.97 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.42 (s, 3H); LCMS
(APCI+) m/z 336.2 (M + 1).
1
yield). H NMR (400 MHz, CDCl3-d) δ 7.37 (dd, J1 = 8.6 Hz, J2 =
2.3 Hz, 1H), 6.95 (s, 1H), 6.91 (d, J = 8.6 Hz, 1H), 6.75 (m, 2H), 6.55
(m, 1H), 3.82 (s, 3H), 3.19 (s, 3H), 2.56 (d, J = 14.1 Hz, 1H), 1.97 (d,
J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.41 (s, 3H); LCMS (APCI+) m/z
384.2 (M + 1).
2′-Amino-6-(3-chlorophenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-12). was synthesized in
the same fashion as compound 11, substituting 3-chlorophenylboronic
acid for phenylboronic acid to afford 2′-amino-6-(3-chlorophenyl)-
1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (8 mg,
2′-Amino-6-(2-fluoro-5-methoxyphenyl)-1′,2,2-
trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-19).
rac-19 was synthesized in the same fashion as compound 11, replacing
phenylboronic acid with (2-fluoro-5-methoxyphenyl)boronic acid to
afford 2′-amino-6-(2-fluoro-5-methoxyphenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (15 mg, 0.039 mmol, 26%
1
0.022 mmol, 29% yield). H NMR (400 MHz, CDCl3-d) δ 7.35 (m,
5H), 6.93 (m, 2H), 3.20 (s, 3H), 2.57 (d, J = 14.1 Hz, 1H), 1.99 (d, J =
14.1 Hz, 1H), 1.52 (s, 3H), 1.40 (s, 3H); LCMS (APCI+) m/z 370.2
(M + 1)
3-(2′-Amino-1′,2,2-trimethyl-5′-oxo-1′,5′-dihydrospiro-
[chroman-4,4′-imidazole]-6-yl)benzonitrile (rac-13). rac-13 was
synthesized in the same fashion as compound 11, replacing
phenylboronic acid with 3-cyanophenylboronic acid to afford 3-(2′-
amino-1′,2,2-trimethyl-5′-oxo-1′,5′-dihydrospiro[chroman-4,4′-imida-
zole]-6-yl)benzonitrile (0.031 g, 0.0860 mmol, 58.2% yield). 1H NMR
(400 MHz, CDCl3-d) δ 7.71 (s, 1H), 7.66 (d, J1 = 7.83 Hz 1H), 7.56
(d, J1 = 7.83 Hz 1H), 7.48 (t, J1 = 7.63 Hz 1H), 7.38 (dd, J1 = 8.61 Hz,
J2 = 1.97 Hz, 1H), 6.96 (d, J1 = 8.61 Hz, 1H), 6.93 (s, 1H), 3.23 (s,
3H), 2.55 (d, J1 = 13.7 Hz, 1H), 1.99 (d, J1= 14.1 Hz, 1H), 1.53 (s,
3H), 1.44 (s, 3H); LCMS (APCI+) m/z 361.1 (M + 1)
1
yield). H NMR (400 MHz, CDCl3) δ 7.37 (m, 1H), 6.99 (m, 2H),
6.91 (d, J = 8.6 Hz, 1H), 6.83 (m, 1H), 6.76 (m, 1H), 3.79 (s, 3H),
3.16 (s, 3H), 2.56 (d, J = 14.1 Hz, 1H), 1.97 (d, J = 14.1 Hz, 1H), 1.52
(s, 3H), 1.41 (s, 3H); LCMS (APCI+) m/z 384.2 (M + 1).
2′-Amino-6-(3-chloro-5-fluorophenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-20). rac-20 was synthe-
sized in the same fashion as compound 11, replacing phenylboronic
acid with (3-chloro-5-fluorophenyl)boronic acid to afford 2′-amino-6-
(3-chloro-5-fluorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imida-
1
zol]-5′(1′H)-one (25 mg, 0.064 mmol, 44% yield). H NMR (400
MHz, CDCl3-d) δ 7.35 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.21 (m,
1H), 7.01 (m, 2H), 6.92 (m, 2H), 3.20 (s, 3H), 2.54 (d, J = 14.1 Hz,
1H), 1.96 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.41 (s, 3H); LCMS
(APCI+) m/z 388.2 (M + 1).
2′-Amino-6-(3-methoxyphenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-14). rac-14 was synthe-
sized in the same fashion as compound 11, substituting 3-
methoxyphenylboronic acid for phenylboronic acid to afford 2′-
L
dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2′-Amino-6-(5-chloro-2-fluorophenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-21). rac-21 was synthe-
sized in the same fashion as compound 11, replacing phenylboronic
acid with (3-chloro-6-fluorophenyl)boronic acid to afford 2′-amino-6-
(5-chloro-2-fluorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imida-
6-(3-fluorophenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-
1
5′(1′H)-one (8 mg, 0.023 mmol, 31% yield). H NMR (400 MHz,
DMSO-d6) δ 7.45 (m, 2H), 7.30 (m, 2H), 7.12 (m, 1H), 6.88 (m,
2H), 3.05 (s, 3H), 2.2 (d, J = 14.1 Hz, 1H), 1.80 (d, J = 14.1 Hz, 1H),
1.43 (s, 3H), 1.39 (s, 3H); LCMS (APCI+) m/z 354.2 (M + 1).
2′-Amino-6-cyclohexyl-1′,2,2-trimethylspiro[chroman-4,4′-
imidazol]-5′(1′H)-one (rac-28). A mixture of 2′-amino-6-bromo-
1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (0.050 g,
0.148 mmol), cyclohexylzinc(II) bromide (0.44 mmol), and bis(tri-
tert-butylphosphine)palladium(0) (0.014 mmol) in THF (1 mL) was
heated in a sealed vial overnight at 90 °C. MS showed that the reaction
was completed. The sample was then purified on silica gel, eluting with
2−10% MeOH/DCM + 1% NH4OH to give 2′-amino-6-cyclohexyl-
1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (13.7 mg,
1
zol]-5′(1′H)-one (25 mg, 0.064 mmol, 44% yield). H NMR (400
MHz, CDCl3-d) δ 7.34 (dm, J1 = 8.59 Hz, J2 = 2.05 Hz, 1H) 7.29 (dd,
J1 = 6.83 Hz, J2 = 2.73 Hz, 1H) 7.20 (m, 1H), 7.02 (dd, J1 = 9.96 Hz, J2
= 8.78 1H), 6.95 (s, 1H), 6.92 (d, J1 = 8.59 Hz, 1H), 3.18 (s, 3H), 2.56
(d, J1 = 14.3 Hz, 1H), 1.98 (d, J1 = 14.3 Hz, 1H), 1.52 (s, 3H), 1.42 (s,
3H); LCMS (APCI+) m/z 388.2 (M + 1).
2′-Amino-6-(3-isopropoxyphenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-22). rac-22 was synthe-
sized in the same fashion as compound 11, replacing phenylboronic
acid with (3-isopropoxyphenyl)boronic acid to afford 2′-amino-6-(3-
isopropoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-
1
0.041 mmol, 27%). H NMR (400 MHz, CDCl3-d) δ 7.04 (dd, J1 =
8.59 Hz, J2 = 2.15 Hz, 1H), 6.76 (d, J1 = 8.40, 1H), 6.61 (d, J1 = 1.95
Hz, 1H), 3.20 (s, 3H), 2.53 (d, J1 = 14.1 Hz, 1H), 2.34 (m, 1H), 1.94
(d, J1 = 14.3 Hz, 1H), 1.75 (m, 5H), 1.47 (s, 3H), 1.35 (s, 3H), 1.25
(m, 5H), LCMS (APCI+) m/z 342.2 (M + 1).
1
5′(1′H)-one (27 mg, 0.069 mmol, 46% yield). H NMR (400 MHz,
CDCl3-d) δ 7.39 (dd, J1 = 8.6 Hz, J2 = 2.3 Hz, 1H), 7.27 (t, J = 7.8 Hz,
1H), 6.99 (m, 3H), 6.90 (d, J = 8.6 Hz, 1H), 6.81 (dd, J1 = 8.2 Hz, J2 =
2.3 Hz, 1H), 4.58 (qn, J = 5.9 Hz, 1H), 3.19 (s, 3H), 2.56 (d, J = 14.1
Hz, 1H), 1.97 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.41 (s, 3H), 1.35 (s,
3H), 1.34 (s, 3H); LCMS (APCI+) m/z 394.2 (M + 1).
2′-Amino-6-isobutoxy-1′,2,2-trimethylspiro[chroman-4,4′-
imidazol]-5′(1′H)-one (rac-29). Step A. A mixture of 2′-amino-6-
bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (2.0
g, 5.91 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaboro-
lane) (9.0 g, 35.5 mmol), Pd(PPh3)4 (0.683 g, 0.59 mmol), 3 M KHF2
(5 mL, 15 mmol), and 2 M Na2CO3 (5.91 mL, 11.8 mmol) in dioxane
(9 mL, 5.91 mmol) was heated in a sealed vial at 90 °C overnight. The
mixture was cooled to ambient temperature, filtered through a Celite
plug, and the filtrate collected was concentrated in vacuo. The residue
obtained was purified by flash chromatography on silica gel, eluting
with 1−10% MeOH/EtOAc + 1% TEA to provide (rac)-2′-amino-
1′,2,2-trimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)spiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (1.65 g, 72%) as a light yellow
2′-Amino-6-(3-ethoxyphenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-23). rac-23 was synthe-
sized in the same fashion as compound 11, replacing phenylboronic
acid with (3-ethoxyphenyl)boronic acid to afford 2′-amino-6-(3-
ethoxyphenyl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-
one (11 mg, 0.029 mmol, 20% yield). 1H NMR (400 MHz, CDCl3-d)
δ 7.34 (dd, J1 = 8.6 Hz, J2 = 2.0 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 6.99
(m, 3H), 6.90 (d, J = 8.6 Hz, 1H), 6.81 (dd, J1 = 8.2 Hz, J2 = 2.3 Hz,
1H), 4.06 (q, J = 7.0 Hz, 2H), 3.19 (s, 3H), 2.56 (d, J = 14.1 Hz, 1H),
1.97 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H), 1.42 (t, J = 7.0 Hz, 3H), 1.41
(s, 3H); LCMS (APCI+) m/z 380.2 (M + 1).
1
solid contaminated with a small amount of dioxaboralane. H NMR
(400 MHz, CDCl3-d) δ 7.64 (dd, J1 = 8.589 Hz, J2 = 1.562 Hz, 1H),
7.29 (br s, 1H), 6.83 (d, J = 8.21 Hz, 1H), 3.21 (s, 3H), 2.55 (d, J =
14.445 Hz, 1H), 1.96 (d, J = 14.055 Hz, 1H), 1.49 (s, 3H), 1.36 (s,
3H), 1.29 (s, 12H); LCMS (APCI+) m/z 286.2 (M + 1), retention
time 2.19 min.
2′-Amino-1′,2,2-trimethyl-6-(3-(trifluoromethyl)phenyl)-
spiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-24). rac-24 was
synthesized in the same fashion as compound 11, replacing
phenylboronic acid with 3-(triflouromethyl)phenylboronic acid to
afford 2′-amino-1′,2,2-trimethyl-6-(3-(trifluoromethyl)phenyl)spiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (13 mg, 0.032 mmol, 22%
Step B. To a solution of 2′-amino-1′,2,2-trimethyl-6-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)spiro[chroman-4,4′-imidazol]-
5′(1′H)-one (0.25 g, 0.649 mmol) in MeOH (5 mL) and CH2Cl2 at 0
°C was added 50% hydrogen peroxide in H2O (1.1 mL, 16.2 mmol).
The reaction mixture was stirred at 0 °C for 45 min and then diluted
with EtOAc/H2O, and the layers were separated. The aqueous layer
was extracted with EtOAc (2×). The combined organic layers were
dried (Na2SO4), filtered, and concentrated in vacuo to provide 2′-
amino-6-hydroxy-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-
5′(1′H)-one (0.113 g, 0.410 mmol, 63.3% yield) as a white foamy
1
yield). H NMR (400 MHz, CDCl3-d) δ 7.66 (s, 1H), 7.60 (d, J1=
7.61 Hz, 1H), 7.54 (d, J1 = 7.61 Hz, 1H), 7.49 (d, J1 = 7.61 Hz, 1H),
7.40 (dd, J1 = 8.59 Hz, J2 = 2.15 Hz, 1H), 6.97 (s, 1H), 6.95 (d, J1 =
8.59 Hz, 1H), 3.19 (s, 3H), 2.56 (d, J1 = 14.1 Hz, 1H), 1.98 (d, J1 =
14.1 Hz, 1H), 1.52 (s, 3H), 1.42 (s, 3H); LCMS (APCI+) m/z 404.2
(M + 1).
2′-Amino-1′,2,2-trimethyl-6-(3-(methylthio)phenyl)spiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-25). rac-25 was synthe-
sized in the same fashion as compound 11, replacing phenylboronic
acid with (3-(methylthio)phenylboronic acid to afford 2′-amino-1′,2,2-
trimethyl-6-(3-(methylthio)phenyl)spiro[chroman-4,4′-imidazol]-
1
solid. H NMR (400 MHz, DMSO-d6) δ 10.20 (s, 1H), 8.789 (brs,
1H), 6.55 (m, 1H), 6.11 (s, 1H), 3.92 (s, 2H), 3.02 (s, 3H), 2.14 (d, J
= 13.66 Hz, 1H), 1.77 (d, J = 12.664 Hz, 1H), 1.36 (s, 3H), 1.29 (s,
3H); LCMS (APCI+) m/z 276.2 (M + 1), retention time 0.78 min.
Step C. To a solution of 2′-amino-6-hydroxy-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (18 mg, 0.0654 mmol) in DMF
(0.5 mL) were added Cs2CO3 (21.3 mg, 0.0654 mmol) and 1-bromo-
2-methylpropane (0.00782 mL, 0.0719 mmol). The reaction mixture
was stirred at ambient temperature. After 18 h, an additional 3 equiv of
Cs2CO3 was added, stirred at room temperature for 2 h, and then
heated at 60 °C overnight. The mixture was cooled to ambient
temperature and partitioned between brine and EtOAc. The combined
aqueous layers were back-extracted with EtOAc. The combined
organic layers were dried (Na2SO4), filtered, and concentrated in
vacuo. The residue obtained was purified by flash chromatography on
silica gel (10 g Snap cartridge, on Biotage SP1) using a gradient of 2−
15% MeOH/CH2Cl2 + 2% NH4OH to provide 2′-amino-6-isobutoxy-
1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (6.2 mg,
1
5′(1′H)-one (23 mg, 0.0619 mmol, 42% yield). H NMR (400 MHz,
CDCl3-d) δ 7.38 (dd, J1 = 8.59 Hz, J2 = 2.15 Hz, 1H), 7.31 (s, 1H),
7.28 (d, J1 = 7.61 Hz, 1H), 7.26 (s, 1H), 7.19 (s, 1H), 7.17 (s, 1H),
6.96 (d, J1 = 1.95 Hz, 1H), 6.91 (d, J1 = 8.59 Hz, 1H), 3.17 (s, 3H),
2.56 (d, J1 = 14.25 Hz, 1H), 2.49 (s, 3H), 1.97 (d, J1 = 14.25 Hz, 1H),
1.51 (s, 3H), 1.41 (s, 3H); LCMS (APCI+) m/z 382.1 (M + 1).
2′-Amino-6-(3-(difluoromethoxy)phenyl)-1′,2,2-
trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-26).
rac-26 was synthesized in the same fashion as compound 11, replacing
phenylboronic acid with (3-(difluoromethoxy)phenyl)boronic acid to
afford 2′-amino-6-(3-(difluoromethoxy)phenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (11 mg, 0.03 mmol, 19% yield).
1H NMR (400 MHz, CDCl3) δ 7.36 (m, 2H), 7.27 (m, 1H), 7.18 (m,
1H), 7.03 (m, 1H), 6.94 (m, 1H), 6.53 (t, J = 74 Hz, 1H), 3.19 (s,
3H), 2.54 (d, J = 14.1 Hz, 1H), 1.99 (d, J = 14.1 Hz, 1H), 1.52 (s, 3H),
1.42 (s, 3H); LCMS (APCI+) m/z 402.1 (M + 1)
2′-Amino-6-(3-fluorophenyl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-27). rac-27 was synthe-
sized in the same fashion as compound 11 substituting 3-
fluorophenylboronic acid for phenylboronic acid to afford 2′-amino-
1
28.6%) as a pale yellow foamy solid. H NMR (400 MHz, CDCl3-
d) δ 6.78−6.77 (m, 2H), 6.37−6.36 (m, 1H), 3.59−3.58 (m, 2H), 3.19
(s, 3H), 2.52 (d, J = 14.09 Hz, 1H), 2.04−1.96 (m, 1H), 1.94 (d, J =
14.09 Hz, 1H), 1.47 (s, 3H), 1.34 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H).
M
dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2′-Amino-1′,2,2-trimethyl-6-(piperidin-1-yl)spiro[chroman-
4,4′-imidazol]-5′(1′H)-one formate (rac-30). A solution of 1 N
lithium bis(trimethylsilyl)amide in THF (1.892 mL, 1.892 mmol) and
piperidine (0.07018 mL, 0.7096 mmol) was injected into a vial
containing Pd2dba3 (10.83 mg, 0.011 83 mmol), 2′-(dicyclohexylphos-
phino)-N,N-dimethylbiphenyl-2-amine (5.585 mg, 0.014 19 mmol),
and 2′-amino-6-bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-
5′(1′H)-one (200 mg, 0.5914 mmol). The mixture was stirred at 65
°C for 15 h. The mixture was cooled to room temperature and
concentrated in vacuo. The residue obtained was purified first by silica
gel chromatography, eluting with a gradient of 10−100% MeOH/
DCM containing 1% NH4OH, then by C-18 reverse phase
chromatography (10−100% CH3CN/H2O (both eluents with 0.5%
TFA) to provide 2′-amino-1′,2,2-trimethyl-6-(piperidin-1-yl)spiro-
[chroman-4,4′-imidazol]-5′(1′H)-one formate (0.8 mg, 0.002 059
1.83 (d, J1 = 14.1 Hz, 1H), 1.43 (s, 3H), 1.39 (s, 3H); MS (APCI/ESI,
multimode) m/z 337.2. (M + 1).
2′-Amino-6-(5-chloropyridin-3-yl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-34). rac-34 was synthe-
sized in the same fashion as compound 11, replacing phenylboronic
acid with 5-chloropyridin-3-ylboronic acid to afford 2′-amino-6-(5-
chloropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-
1
5′(1′H)-one (25 mg, 0.067 mmol, 46% yield). H NMR (400 MHz,
CDCl3-d) δ 8.57 (d, J1 = 1.95 Hz, 1H), 8.48, (d, J1 = 2.34 Hz, 1H),
7.71 (t, J1 = 2.15 Hz, 1H), 7.37 (dd, J1 = 8.59 Hz, J2 = 2.34 Hz, 1H),
6.97 (d, J1 = 8.59 Hz, 1H), 6.94 (s, 1H), 3.21 (s, 3H), 2.55 (d, J1 =
14.1 Hz, 1H), 1.97 (d, J1 = 14.1 Hz, 1H), 1.53 (s, 3H), 1.43 (s, 3H);
LCMS (APCI+) m/z 371.2 (M + 1).
2′-Amino-1′,2,2-trimethyl-6-(5-(trifluoromethyl)pyridin-3-
yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-35). rac-35
was synthesized in the same fashion as compound 11, replacing
phenylboronic acid with 5-(trifluoromethyl)pyridin-3-ylboronic acid to
afford 2′-amino-1′,2,2-trimethyl-6-(5-(trifluoromethyl)pyridin-3-yl)-
spiro[chroman-4,4′-imidazol]-5′(1′H)-one (33 mg, 0.082 mmol, 55%
1
mmol, 0.35% yield). H NMR (400 MHz, CD3OD-d4) δ 6.98 (dd,
J1 = 8.98 Hz, J2 = 2.73 Hz, 1H), 6.76 (d, J1 = 8.98 Hz, 1H), 6.49 (d, J1
= 2.73 1H), 3.23 (s, 3H), 2.92 (t, J1 = 4.57 Hz, 4H), 2.37 (d, J1 = 14.4
Hz, 1H), 2.14 (d, J1 = 14.4 Hz, 1H), 1.67 (m, 4H), 1.51 (m, 2H), 1.43
(s, 3H), 1.32 (s, 3H); MS (APCI+/ESI+ multimode) m/z 343.2 (M +
1).
1
yield). H NMR (400 MHz, CDCl3-d) δ 8.88 (s, 1H), 8.79 (s, 1H),
7.94 (s, 1H), 7.41 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz, 1H), 7.0 (d, J1 =
8.61 Hz, 1H), 6.97 (d, J1 = 1.96 Hz, 1H), 3.20 (s, 3H), 2.55 (d, J1 =
14.1 Hz, 1H), 1.98 (d, J1 = 14.1 Hz, 1H), 1.54 (s, 3H), 1.44 (s, 3H);
LCMS (APCI+) m/z 405.2 (M + 1).
2′-Amino-1′,2,2-trimethyl-6-(pyridin-3-yl)spiro[chroman-
4,4′-imidazol]-5′(1′H)-one (rac-31). rac-31 was synthesized in the
same fashion as compound 11, replacing phenylboronic acid with
pyridin-3-ylboronic acid to afford 2′-amino-1′,2,2-trimethyl-6-(pyridin-
3-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (18 mg, 0.05351
2′-Amino-1′,2,2-trimethyl-6-(5-(prop-1-ynyl)pyridin-3-yl)-
spiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-36). A resealable
glass pressure tube was charged with 2′-amino-1′,2,2-trimethyl-6-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)spiro[chroman-4,4′-imi-
dazol]-5′(1′H)-one (30 mg, 0.078 mmol), 3-bromo-5-(prop-1-ynyl)-
pyridine (18 mg, 0.093 mmol), dichloro[1,1′-bis(diphenylphosphino)-
ferrocene]palladium(II) dichloromethane adduct (3.2 mg, 0.0039
mmol), 20% aqueous Na2CO3 (144 μL, 0.27 mmol), and 1,4-dioxane
(779 μL, 0.078 mmol). The mixture was sparged with N2 for 5 min,
capped, and stirred at 90 °C for 1 h. The solvents were removed in
vacuo, and the residue obtained was suspended in 1.5 mL of MeOH
and filtered through a 45 μm filter. The filtrate collected was purified
by C-18 reverse phase preparative HPLC on Gilson UniPoint
instrument using a 5−95% CH3CN/water + 0.5% formic acid
gradient. The fractions containing the product were pooled,
concentrated in vacuo, evaporated from CH3CN (2 × 5 mL), and
dried under high vacuum for 48 h to give 2′-amino-1′,2,2-trimethyl-6-
(5-(prop-1-ynyl)pyridin-3-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-
one (23 mg, 0.061 mmol, 79% yield). 1H NMR (400 MHz, CDCl3-d)
δ 8.55 (d, J1 = 10.96 Hz, 2H), 7.72 (s, 1H), 7.42 (dd, J1 = 8.61 Hz, J2 =
1.96 Hz, 1H), 6.99 (d, J1 = 8.61 Hz, 1H), 6.95 (d, J1 = 2.35 Hz, 1H),
3.30 (s, 3H), 2.57 (d, J1 = 14.5 Hz, 1H), 2.12 (d, J1 = 14.5 Hz, 1H),
2.09 (s, 3H), 1.54 (s, 3H), 1.46 (s, 3H); LCMS (APCI+) m/z 375.2
(M + 1).
1
mmol, 24.13% yield). H NMR (400 MHz, DMSO-d6) δ 8.69 (d, J1
= 1.96 Hz, 1H), 8.50 (dd, J1 = 4.70 Hz, J2 = 1.57 Hz, 1H), 7.87 (dt, J1
= 8.61 Hz, J2 = 1.96 Hz 1H), 7.49 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz, 1H),
7.42 (dd, J1 = 7.83 Hz, J2 = 4.70 Hz, 1H), 6.94 (d, J1 = 2.35 Hz 1H),
6.89 (d, J1 = 8.61 Hz, 1H), 6.52 (s, 2H), 3.04 (s, 3H), 2.22 (d, J1 =
14.1 Hz, 1H), 1.84 (d, J1 = 13.7 Hz, 1H), 1.46 (s, 3H), 1.39 (s, 3H);
MS (APCI+) m/z 337.1 (M + 1).
2′-Amino-1′,2,2-trimethyl-6-(pyridin-2-yl)spiro[chroman-
4,4′-imidazol]-5′(1′H)-one (rac-32). Step A. A mixture of 2′-amino-
6-bromo-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one
(2.0 g, 5.91 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxa-
borolane) (9.01 g, 35.5 mmol), Pd(PPh3)4 (0.683 g, 0.591 mmol), and
Na2CO3 (5.91 mL, 11.8 mmol) in dioxane (9 mL, 5.91 mmol) was
heated in a sealed vial overnight at 90 °C. The mixture was filtered
through Celite 545 and concentrated in vacuo. The residue obtained
was purfied on silica gel, eluting with 1−10% MeOH/EtOAc (1%
TEA) to provide 2′-amino-1′,2,2-trimethyl-6-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one
(1.65 g, 72.4%).
Step B. A solution of 4:1 dioxane/water (1 mL) was sparged with
N2 for 30 min. The sparged solution was added to a pressure vessel
containing 2′-amino-1′,2,2-trimethyl-6-(4,4,5,5-tetramethyl-1,3,2-diox-
aborolan-2-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (100 mg,
0.2596 mmol), 2-chloropyridine (0.036 54 mL, 0.3893 mmol),
Pd(PPh3)4 (29.99 mg, 0.025 96 mmol), and 2 M Na2CO3 (82.53
mg, 0.7787 mmol). The contents were sealed and heated to 80 °C for
15 h. The mixture was concentrated and purified by silica gel
chromatography, eluting with a gradient of 0−30% MeOH/EtOAc
containing 6% NH4OH to provide 2′-amino-1′,2,2-trimethyl-6-
(pyridin-2-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (18 mg,
0.05351 mmol, 20.62% yield) as an off-white powder. 1H NMR
(400 MHz, DMSO-d6) δ 8.59 (d, J1 = 4.70 Hz, 1H), 7.80 (m, 3H),
7.42 (s, 1H), 7.25 (m, 1H), 6.88 (d, J1 = 8.61 Hz, 1H), 6.55 (s, 2H),
3.05 (s, 3H), 2.23 (d, J1 = 13.3 Hz, 1H), 1.83 (d, J1 = 14.5 Hz 1H),
1.47 (s, 3H), 1.39 (s, 3H); MS (APCI+) m/z 337.1 (M + 1).
2′-Amino-1′,2,2-trimethyl-6-(pyridin-4-yl)spiro[chroman-
4,4′-imidazol]-5′(1′H)-one (rac-33). rac-33 was synthesized in the
same fashion as compound 11, replacing phenylboronic acid with
(pyridin-4-ylboronic acid to afford 2′-amino-1′,2,2-trimethyl-6-(pyr-
idin-4-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (18 mg, 0.05351
2′-Amino-6-(2-fluoropyridin-3-yl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-37). rac-37 was synthe-
sized in the same fashion as compound 32, substituting 2-
chloropyridine for 2-fluoro-3-iodopyridine to afford 2′-amino-6-(2-
fluoropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-
1
5′(1′H)-one (15 mg, 0.04233 mmol, 32.61% yield). H NMR (400
MHz, DMSO-d6) δ 8.13 (d, J1 = 7.70 Hz, 1H), 7.90 (m, 1H), 7.40 (d,
J1 = 8.61 Hz 1H), 7.35 (m, 1H), 7.05 (s, 1H), 6.91 (d, J1 = 8.61 Hz,
1H), 2.79 (s, 3H), 2.36 (d, J1 = 11.74 Hz, 1H), 1.89 (m, 1H), 1.47 (s,
3H), 1.45 (s, 3H); MS (APCI+/ESI+ multimode) m/z 55.2 (M + 1).
2′-Amino-6-(5-fluoropyridin-3-yl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-38). rac-38 was synthe-
sized in the same fashion as compound 11, replacing phenylboronic
acid with 5-fluoropyridin-3-yl boronic acid (21 mg, 0.15 mmol) to
afford 2′-amino-6-(5-fluoropyridin-3-yl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (10 mg, 0.028 mmol, 19%
1
yield). H NMR (400 MHz, CDCl3-d) δ 8.53 (s, 1H), 8.38 (d, J1 =
1
2.54 Hz, 1H), 7.44 (dt, J1 = 9.57 Hz, J2 = 2.25 Hz, 1H), 7.38 (dd, J1 =
8.59 Hz, J2 = 2.34 Hz, 1H), 6.97 (d, J1 = 8.59 Hz, 1H), 6.96 (s, 1H),
3.20 (s, 3H), 2.55 (d, J1 = 14.1 Hz, 1H), 1.97 (d, J1 = 14.3 Hz, 1H),
1.53 (s, 3H), 1.43 (s, 3H); LCMS (APCI+) m/z 355.2 (M + 1).
mmol, 18.1% yield). H NMR (400 MHz, DMSO-d6) δ 8.55 (d, J1 =
6.26 Hz 2H), 7.59 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz 1H), 7.52 (dd, J1 =
4.70 Hz, J2 = 1.57 Hz, 2H), 7.02 (d, J1 = 2.35 Hz 1H), 6.92 (d, J1 =
8.61 Hz 1H), 6.55 (s, 2H), 3.05 (s, 3H), 2.22 (d, J1 = 14.1 Hz, 1H),
N
dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2′-Amino-1′,2,2-trimethyl-6-(pyrimidin-5-yl)spiro[chroman-
4,4′-imidazol]-5′(1′H)-one (rac-39). rac-39 was synthesized in the
same fashion as compound 11, replacing phenylboronic acid with
pyrimidin-5-yl boronic acid to afford 2′-amino-1′,2,2-trimethyl-6-
(pyrimidin-5-yl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (0.037 g,
KCN were added to the mixture. The bomb was sealed and heated
back to 150 °C for a further 24 h. The bomb mixture was cooled to
ambient temperature, and the contents were poured into water. The
rapidly stirred suspension was carefully acidified with 5 M hydrochloric
acid (37.28 mL, 186.4 mmol). The mixture was diluted further with
water (50 mL), and the resulting solids were filtered, washing with
water. The solid collected was first air-dried and then vacuum-dried for
24 h to provide 6′-bromo-2′,2′-dimethylspiro[imidazolidine-4,4′-
1
0.110 mmol, 74.2% yield). H NMR (400 MHz, CDCl3-d) δ 9.13 (s,
1H), 8.83 (s, 2H), 7.39 (d, J1 = 6.65 Hz, 1H), 7.00 (d, J1 = 8.61 Hz,
1H), 6.95 (s, 1H), 3.19 (s, 3H), 2.54 (d, J1 = 14.5 Hz, 1H), 1.97 (d, J1
= 14.1 Hz, 1H), 1.53 (s, 3H), 1.44 (s, 3H); LCMS (APCI+) m/z
338.3 (M + 1).
1
thiochroman]-2,5-dione (1.8 g, 7.75% yield). H NMR (400 MHz,
CDCl3-d) δ 7.34−7.32 (m, 2H), 7.05 (J = 8.61 Hz, 1H), 2.68 (d, J =
14.08 Hz, 1H), 2.29 (d, J = 14.86 Hz, 1H), 1.47 (s, 3H), 1.45 (s, 3H).
MS (neg) m/z 339.0, 341.0 (M − 1).
2′-Amino-6-(isothiazol-5-yl)-1′,2,2-trimethylspiro[chroman-
4,4′-imidazol]-5′(1′H)-one (rac-40). A glass pressure tube was
charged with a mixture of 2′-amino-6-bromo-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (50 mg, 0.15 mmol), 5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)isothiazole (62 mg, 30 mmol),
Pd(PPh3)4 (8.5 mg, 0.0074 mmol), 2 M Na2CO3 (0.15 mL, 0.30
mmol), and dioxane (1.5 mL, 0.15 mmol). The tube was sealed and
heated at 90 °C for 18 h. The crude reaction mixture was directly
purified by flash chromatography on silica gel, eluting with a gradient
of 2−10% MeOH/DCM containing 1% NH4OH to provide 2′-amino-
6-(isothiazol-5-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-
5′(1′H)-one (9 mg, 18%) as a white solid. 1H NMR (400 MHz,
CDCl3-d) δ 8.395 (d, J = 1.56 Hz, 1H), 7.414 (dd, J1 = 8.589 Hz, J2 =
1.56 Hz, 1H), 7.26 (s, 1H), 7.22 (d, J = 1.952 Hz, 1H), 6.91 (d, J =
8.589 Hz, 1H), 3.21 (s, 3H), 2.54 (d, 14.06 Hz, 1H), 1.96 (d, J = 14.45
Hz, 1H), 1.52 (s, 3H), 1.42 (s, 3H); LCMS (APCI+) m/z 343.1 (M +
1), retention time 1.996 min.
N-(2′-Amino-1′,2,2-trimethyl-5′-oxo-1′,5′-dihydrospiro-
[chroman-4,4′-imidazole]-6-yl)-5-methylfuran-2-carboxamide
(rac-41). Step A. A mixture of 2′-amino-6-bromo-1′,2,2-
trimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (2.0 g, 5.91
mmol), tert-butyl carbamate (6.93 g, 59.1 mmol), and Cs2CO3 (3.85
g, 11.8 mmol) in THF (20 mL, 5.91 mmol) was purged with Ar for 5
min. Pd2dba3 (542 mg, 0.591 mmol) and XPHOS (564 mg, 1.18
mmol) were added, and the mixture was heated to 90 °C overnight in
a sealed vial. The mixture was then cooled to ambient temperature and
filtered through Celite. The filtrate collected was concentrated in
vacuo, and the residue obtained was purified by flash chromatography
on silica gel, eluting with CH2Cl2/MeOH/NH4OH (90:9:1). The
product isolated was taken up in DCM/TFA (1:3, 20 mL) and stirred
at ambient temperature for 3 h. The mixture was concentrated in
vacuo, and the resulting oil was evaporated from EtOAc and hexanes
to provide 2′,6-diamino-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-
5′(1′H)-one (1.15 g, 71% yield) as a light brown solid which was used
for the next reaction directly. LCMS (APCI+) m/z 275.1 (M + 1).
Step B. To a mixture of 2′,6-diamino-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (0.032 g, 0.0862 mmol) and 5-
methylfuran-2-carbonyl chloride (0.0125 g, 0.0862 mmol) in DCM
(0.007 32 g, 0.0862 mmol) was added TEA (d, 0.726) (0.0120 mL,
0.0862 mmol), and the mixture was stirred for 20 min at room
temperature. The mixture was concentrated in vacuo, and the residue
obtained was purified on reverse phase C-18 HPLC to give N-(2′-
amino-1′,2,2-trimethyl-5′-oxo-1′,5′-dihydrospiro[chroman-4,4′-imida-
zole]-6-yl)-5-methylfuran-2-carboxamide (15 mg, 0.0384 mmol, 45%
yield). 1H NMR (400 MHz, CDCl3-d) δ 11.72 (br s, 1H), 11.09 (br s,
1H), 8.14 (s, 1H), 7.55 (s, 1H), 7.35 (d, J = 9.39 Hz, 1H), 7.06 (d, J =
3.13 Hz, 1H), 6.89 (d, J = 8.61 Hz, 1H), 6.15 (d, J = 3.13 Hz, 1H),
3.30 (s, 3H), 2.56 (d, J = 14.86 Hz, 1H), 2.37 (s, 3H), 2.17 (d, J =
14.08 Hz, 1H), 1.52 (s, 3H), 1.39 (s, 3H). LCMS (APCI+) m/z 483.2
(M + 1), retention time 1.956 min.
Step B. 6′-Bromo-2′,2′-dimethylspiro[imidazolidine-4,4′-thiochro-
man]-2,5-dione (1.75 g, 5.13 mmol) in DMF (0.4 M, 9.8 mL) was
treated with K2CO3 (709 mg, 5.13 mmol) followed by iodomethane
(0.319 mL, 5.13 mmol) at ambient temperature. After 30 min, the
reaction was diluted with water (50 mL), and the resulting solid was
filtered, air-dried, and dried under high vacuum for 16 h to provide 6′-
bromo-1,2′,2′-trimethylspiro[imidazolidine-4,4′-thiochroman]-2,5-
dione (1.63 g, 4.59 mmol, 89.5% yield) as a white solid. 1H NMR (400
MHz, CDCl3-d) δ 7.35−7.32 (m, 1H), 7.18−7.17 (m, 1H), 7.07 (dd,
J1 = 8.61 Hz, J2 = 2.35 Hz, 1H), 3.17 (s, 3H), 2.68 (d, J = 14.86 Hz,
1H), 2.12 (d, J = 14.86 Hz, 1H), 0.01 (s, 6H); MS (neg) m/z 352.1,
354.9 (M − 1)−
.
Step C. 6′-Bromo-1,2′,2′-trimethylspiro[imidazolidine-4,4′-thio-
chroman]-2,5-dione (1.60 g, 4.50 mmol) in THF was concentrated
to a paste. The paste was suspended in toluene (9 mL, 0.5 M) and
heated to reflux under a reflux condenser fitted with a drying tube.
When the mixture became a solution, Lawesson’s reagent (1.09 g, 2.70
mmol) was added and heating at reflux was continued for 16 h. The
mixture was cooled to ambient temperature and partitioned between
EtOAc and water. The aqueous layer was extracted a second time with
ethyl acetate. The combined organics were dried (Na2SO4), filtered,
and concentrated in vacuo. The residue isolated was purified by flash
chromatography on silica gel, eluting with 5−50% ethyl acetate−
hexanes to provide 6′-bromo-1,2′,2′-trimethyl-2-thioxospiro-
[imidazolidine-4,4′-thiochroman]-5-one (1.04 g, 2.80 mmol, 62.2%
1
yield) as a white solid. H NMR (400 MHz, CDCl3-d) δ 7.37−7.34
(m, 1H), 7.10−7.06 (m, 2H), 3.39 (s, 3H), 2.62 (d, J = 14.87 Hz, 1H),
2.26 (d, J = 14.87 Hz, 1H), 1.50 (s, 3H), 1.46 (s, 3H); MS (neg) m/z
368.9, 371.1 (M − 1).
Step D. 6′-Bromo-1,2′,2′-trimethyl-2-thioxospiro[imidazolidine-
4,4′-thiochroman]-5-one (35 mg, 0.094 mmol) in DCM (0.5 mL)
was treated with 7 N ammonia in methanol (673 μL, 4.7 mmol) at
ambient temperature. Then 70% tert-butyl hydroperoxide in water (52
μL, 0.38 mmol) was added, and the mixture was stirred at ambient
t
temperature. After 16 h, another 2 equiv of Bu-OOH was added, and
the mixture was stirred at room temperature for another 12 h. The
reaction mixture was transferred to separatory funnel and partitioned
between brine and ethyl acetate. The phases were separated, and the
aqueous layer was extracted with ethyl acetate (1×). The combined
organics were dried (Na2SO4), filtered, concentrated, and purified by
flash chromatography on silica gel, eluting with 4−20% methanol−
dichloromethane to provide 2-amino-6′-bromo-1,2′,2′-trimethylspiro-
[imidazole-4,4′-thiochroman]-5(1H)-one (14 mg, 0.040 mmol, 42%
yield) as a white solid. 1H NMR (400 MHz, CDCl3-d) δ 7.26 (m, 1H),
7.04−7.01 (m, 1H), 3.18 (s, 3H), 2.61−2.56 (m, 1H), 2.09−2.05 (m,
1H), 1.50 (s, 3H), 1.46 (s, 3H); MS (APCI+) m/z 354.2, 356.0 (M +
1).
Step E. 2-Amino-6′-bromo-1,2′,2′-trimethylspiro[imidazole-4,4′-thi-
ochroman]-5(1H)-one (42 mg, 0.119 mmol) in toluene (0.1 M) was
treated with 3-chloro-5-fluorophenylboronic acid (26.9 mg, 0.154
mmol), 20% aqueous sodium carbonate (157 μL, 0.296 mmol), and
tetrakis(triphenylphosphine)palladium(0) (6.85 mg, 0.005 93 mmol).
The mixture was sparged with Ar for 2 min, capped, and heated to
reflux for 16 h. The mixture was applied directly to silica gel, eluting
with a gradient of 4−20% methanol−dichloromethane to provide 2-
amino-6′-(3-chloro-5-fluorophenyl)-1,2′,2′-trimethylspiro[imidazole-
4,4′-thiochroman]-5(1H)-one (30 mg, 0.0743 mmol, 62.7% yield) as a
tan solid. 1H NMR (500 MHz, CDCl3-d) δ 7.33 (dd, J1 = 8.321 Hz, J2
2-Amino-6′ -(3-chloro-5-fluorophenyl)-1, 2′, 2′-
trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one (rac-
42). Step A. 6-Bromo-2,2-dimethylthiochroman-4-one (7.456 mL,
7.456 mmol) in absolute ethanol (1M, 7.4 mL) was treated
sequentially with (NH4)2CO3 (7.165 g, 74.56 mmol), potassium
cyanide (1.457 g, 22.37 mmol), and sodium acid sulfite (0.1552 g,
1.491 mmol) in a steal bomb containing a stir bar. The bomb was
sealed and heated in an oil bath at 150 °C for 16 h. The mixture was
cooled to ambient temperature and saw about >75% conversion by
LC. Therefore an additional 4 equiv of (NH4)2CO3 and 1 equiv of
O
dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
= 1.664 Hz, 1H), 7.25 (m, 2H), 7.04 (m, 3H), 3.22 (s, 3H), 2.65 (d, J
= 14.146 Hz, 1H), 2.14 (d, J = 14.146 Hz, 1H), 1.56 (s, 3H), 1.50 (s,
3H). 13C NMR (500 MHz, CDCl3-d) 155.8 (C), 143.6 (C), 136.0
(C), 135.3 (C), 132.1 (C), 131.9 (C), 131.9 (C), 128.9 (CH), 127.3
(CH), 125.9 (CH), 122.9 (CH), 113 (CH), 113 (CH), 112 (C), 67.4
(C), 49.5 (CH2), 41.4 (C), 31.5−29.2 (CH3), 26.1 (CH3). LCMS
(APCI+) m/z 404.1 (M + 1), retention time = 1.10 min.
1H), 2.61 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.29 (d, J = 14.08 Hz,
1H), 1.82 (dd, J1 = 14.08 Hz, J2 = 2.35 Hz, 1H), 1.17 (s, 3H), 1.02 (s,
3H). MS (positive) m/z 387 (M + 1)+. Analytical HPLC purity = 93%
(220 nm), retention time 3.546 min.
3-(2-Amino-1,2′,2′-trimethyl-5-oxo-1,5-dihydrospiro-
[imidazole-4,4′-thiochroman]-6′-yl)benzonitrile (rac-44). 2-
Amino-6′-bromo-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]-
5(1H)-one (51 mg, 0.144 mmol) and 3-cyanophenylboronic acid
(31.7 mg, 0.216 mmol) were suspended in dioxane (0.75 mL), and
20% aqueous sodium carbonate (237 μL, 0.446 mmol) was added with
rapid stirring. The suspension was sparged with argon for 2 min, at
which point dichloro[1,1′-bis(diphenylphosphino)ferrocene]-
palladium(II) dichloromethane adduct (6 mg, 0.0072 mmol) was
added. The reaction vial was sealed and heated to 100 °C for 16 h. The
reaction mixture was partitioned between EtOAc and water. The
aqueous layer was extracted a second time with ethyl acetate. The
combined organics were dried (Na2SO4), filtered, concentrated, and
purified on silica gel, eluting with 1−15% MeOH/DCM (containing
2% ammonia) to provide 3-(2-amino-1,2′,2′-trimethyl-5-oxo-1,5-
dihydrospiro[imidazole-4,4′-thiochroman]-6′-yl)benzonitrile (32 mg,
0.0850 mmol, 59% yield) as a solid. 1H NMR (400 MHz, CDCl3-d) δ
7.94 (m, 1H), 7.80 (d, J = 1.56 Hz, 1H), 7.79 (m, 1H), 7.65 (t, J = 7.83
Hz, 1H), 7.52−7.49 (m, 1H), 7.19 (d, J = 7.83 Hz, 1H), 6.98 (brs,
1H), 6.54 (brs, 2H), 3.05 (s, 3H), 2.35 (d, J = 14.09 Hz, 1H), 1.97−
1.89 (m, 1H), 1.51 (s, 3H), 1.42 (m, 3H); MS (APCI+) m/z 377.2 (M
+ 1); analytical HPLC purity 95 area %, retention time 3.324 min.
3-(2-Amino-1,3′,3′-trimethyl-5-oxo-1,3′,4′,5-tetrahydro-2′H-
spiro[imidazole-4,1′-naphthalene]-7′-yl)benzonitrile (rac-45).
2-Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2’H-spiro-
[imidazole-4,1′-naphthalen]-5(1H)-one (50 mg, 0.15 mmol) and 3-
cyanophenylboronic acid (28 mg, 0.19 mmol) were processed
according to the method described for the synthesis of 3-(2-amino-
1,2′,2′-trimethyl-5-oxo-1,5-dihydrospiro[imidazole-4,4′-thiochroman]-
6′-yl)benzonitrile to provide 3-(2-amino-1,3′,3′-trimethyl-5-oxo-
1,3′,4′,5-tetrahydro-2′H-spiro[imidazole-4,1′-naphthalene]-7′-yl)-
2-Amino-7′-(3-chloro-5-fluorophenyl)-1,3′,3′-trimethyl-
3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one
(rac-43). Step A. 7-Bromo-3,3-dimethyl-3,4-dihydronaphthalen-
1(2H)-one (3.00 g, 11.9 mmol) in EtOH (12 mL) was treated with
sodium bisulfite (100 mg), KCN (2.32 g, 35.6 mmol), and
(NH4)2CO3 (7.97 g, 83.0 mmol) as described for the synthesis of
6′-bromo-2′,2′-dimethylspiro[imidazolidine-4,4′-thiochroman]-2,5-
dione to provide 7′-bromo-3′,3′-dimethyl-3′,4′-dihydro-2′H-spiro-
[imidazolidine-4,1′-naphthalene]-2,5-dione (1.5 g, 4.64 mmol, 39%
1
yield) as an off-white solid. H NMR (400 MHz, DMSO-d6) δ 11.01
(br s, 1H), 8.58 (s, 1H), 7.46 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz, 1H),
7.14−7.12 (m, 2H), 2.59−2.52 (m, 2H), 2.10 (d, J = 14.09 Hz, 1H),
1.79 (dd, J1 = 14.09 Hz, J2 = 1.57 Hz, 1H), 1.07 (s, 3H), 0.86 (s, 3H).
Step B. 7′-Bromo-3′,3′-dimethyl-3′,4′-dihydro-2′H-spiro-
[imidazolidine-4,1′-naphthalene]-2,5-dione (15.0 g, 46.41 mmol) in
DMF (60 mL) was treated with K2CO3 (6.735 g, 48.73 mmol) and
CH3I (3.04 mL, 48.73 mmol) according to the procedure described for
the synthesis of 6′-bromo-1,2′,2′-trimethylspiro[imidazolidine-4,4′-
thiochroman]-2,5-dione to provide 7′-bromo-1,3′,3′-trimethyl-3′,4′-
dihydro-2′H-spiro[imidazolidine-4,1′-naphthalene]-2,5-dione (15.6 g,
46.26 mmol, 99.67% yield). 1H NMR (CDCl3-d) δ 7.36 (dd, J1 = 8.61
Hz, J2 = 2.35 Hz, 1H), 7.12 (d, J = 2.35 Hz, 1H), 6.99 (d, J = 8.61 Hz,
1H), 6.15 (br s, 1H), 3.13 (s, 3H), 2.72 (s, J = 16.43 Hz, 1H), 2.52
(dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.32 (d, J = 14.09 Hz, 1H), 1.82
(dd, J1 = 14.08 Hz, J2 = 3.13 Hz, 1H), 1.61 (s, 3H), 0.95 (s, 3H).
Analytical HPLC purity 99%, retention time 3.780 min.
Step C. 7′-Bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro-
[imidazolidine-4,1′-naphthalene]-2,5-dione (874 mg, 2.59 mmol)
was treated with Lawesson’s reagent (629 mg, 1.56 mmol), as
described for the synthesis of 6′-bromo-1,2′,2′-trimethyl-2-
thioxospiro[imidazolidine-4,4′-thiochroman]-5-one to provide 7′-
bromo-1,3′,3′-trimethyl-2-thioxo-3′,4′-dihydro-2′H-spiro-
[imidazolidine-4,1′-naphthalen]-5-one (440 mg, 1.25 mmol, 48%
1
benzonitrile (34 mg, 0.095 mmol, 64% yield). H NMR (400 MHz,
CDCl3-d) δ 7.74 (d, J = 1.56 Hz, 1H), 7.69 (d, J = 7.83 Hz, 1H),
7.61−7.58 (m, 1H), 7.50 (t, J = 7.83 Hz, 1H), 7.39 (dd, J1 = 7.83 Hz,
J2 = 2.25 Hz, 1H), 7.21 (d, J = 7.83 Hz, 1H), 7.06 (d, J = 1.56 Hz, 1H),
3.21 (s, 3H), 2.83 (d, J = 15.65 Hz, 1H), 2.61 (dd, J1 = 16.43 Hz, J2 =
2.35 Hz, 1H), 2.29 (d, J = 14.09 Hz, 1H), 1.81 (dd, J1 = 14.09 Hz, J2 =
2.35 Hz, 1H), 1.17 (s, 3H0, 1.02 (s, 3H); LCMS (APCI+) m/z 359
(M + 1), retention time 2.263 min.
1
yield). H NMR (400 MHz, CDCl3-d) δ 7.38 (dd, J1 = 8.61 Hz, J2
= 2.35 Hz, 1H), 7.34 (brs, 1H), 7.02−6.99 (m, 2H), 3.37 (s, 3H), 2.72
(d, J = 16.43 Hz, 1H), 2.53 (dd, J1 = 16.43 Hz, J2 = 3.13 Hz, 1H), 2.27
(d, J = 14.08 Hz, 1H), 1.85 (dd, J1 = 14.08 Hz, J2 = 2.35 Hz, 1H), 1,17
(s, 3H), 0.98 (s, 3H). LCMS (neg) m/z 351, 352 (M − 1); retention
time 1.666 min.
2-Amino-7′-(2-fluoropyridin-3-yl)-1,3′,3′-trimethyl-3′,4′-di-
hydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (rac-
46). 2-Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro-
[imidazole-4,1′-naphthalen]-5(1H)-one (50 mg, 0.15 mmol) and 2-
fluoropyridin-3-ylboronic acid (27 mg, 0.19 mmol) were processed
according to the method described for the synthesis of 2′-amino-6-(2-
fluoropyridin-3-yl)-1′,2,2-trimethylspiro[chroman-4,4′-imidazol]-
5′(1′H)-one to yield 2-amino-7′-(2-fluoropyridin-3-yl)-1,3′,3′-trimeth-
yl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (37
Step D. 7′-Bromo-1,3′,3′-trimethyl-2-thioxo-3′,4′-dihydro-2′H-
spiro[imidazolidine-4,1′-naphthalen]-5-one (2.44 g, 6.91 mmol) in
THF (17 mL) was treated with 7 N NH3 in MeOH (19.7 mL, 138
mmol) and 70% tert-butyl hydroperoxide in water (9.88 mL, 69.1
mmol) as described for the synthesis of 2-amino-6′-bromo-1,2′,2′-
trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one to provide 2-
amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-
4,1′-naphthalen]-5(1H)-one (1.71 g, 5.09 mmol, 73.6% yield) as a
1
mg, 0.10 mmol, 71% yield). H NMR (400 MHz, CDCl3-d) δ 8.16
(dd, J1 = 7.04 Hz, J2 = 1.56 Hz, 1H), 7.80−7.75 (m, 1H), 7.42 (dt, J1 =
7.83 Hz, J2 = 1.56 Hz,1H), 7.26−7.23 (m, 1H), 7.27 (d, J = 7.82 Hz,
1H), 7.10 (s, 1H), 3.20 (s, 3H), 2.82 (d, J = 15.65 Hz, 1H), 2.62 (dd,
J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.31 (d, J = 14.09 Hz, 1H), 1.83 (dd,
J1 = 14.09 Hz, J2 = 3.13 Hz, 1H), 1.17 (s, 3H), 1.03 (s, 3H); 13C NMR
(400 MHz, CDCl3-d) δ 178.4 (C), 160 (C), 156.0 (C), 146.4 (CH),
140.5 (CH), 137.4 (C), 134.2 (C), 132.6 (C), 130.5 (CH), 128.8
(CH), 127.8 (CH), 123.4 (C), 121.8 (CH), 65.6 (C), 46.8 (CH2),
43.4 (CH2), 31.3−25.8 (CH3), 29.6 (C), 25.8 (CH3); LCMS (APCI+)
m/z 353.2 (M + 1), retention time 2.099 min.
5-Amino-6′-(5-chloropyridin-3-yl)-1,2′,2′-trimethylspiro-
[pyrrole-3,4′-thiochroman]-2(1H)-one (rac-47). rac-47 was syn-
thesized in the same fashion as 2-amino-6′-(3-chloro-5-fluorophenyl)-
1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one, substi-
tuting 5-chloropyridin-3-ylboronic acid (14.4 mg, 0.0915 mmol) for 3-
chloro-5-fluorophenylboronic acid to afford 5-amino-6′-(5-chloropyr-
idin-3-yl)-1,2′,2′-trimethylspiro[pyrrole-3,4′-thiochroman]-2(1H)-one
1
white powder. H NMR (400 MHz, CDCl3-d) δ 7.32 (dd, J1 = 7.83
Hz, J2 = 1.56 Hz, 1H), 7.06 (d, J = 1.56 Hz, 1H), 6.97 (d, J = 8.61 Hz,
1H), 3.18 (s, 3H), 2.71 (d, J = 15.65 Hz, 1H), 2.50 (dd, J1 = 16.43 Hz,
J2 = 3.13 Hz, 1H), 2.23 (d, J = 14.08 Hz, 1H), 1.75 (dd, J1 = 14.08 Hz,
J2 = 2.35 Hz, 1H), 1.14 (s, 3H), 0.963 (s, 3H). LCMS (APCI+) m/z
336, 338 (M + 1), retention time 0.983 min.
Step E. 2-Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-
spiro[imidazole-4,1′-naphthalen]-5(1H)-one (56 mg, 0.17 mmol)
and 3-chloro-5-fluorophenylboronic acid (38 mg, 0.22 mmol) were
processed as described for the synthesis of 2-amino-6′-(3-chloro-5-
fluorophenyl)-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]-
5(1H)-one to yield 2-amino-7′-(3-chloro-5-fluorophenyl)-1,3′,3′-tri-
methyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-
one (37 mg, 0.096 mmol, 58% yield). 1H NMR (400 MHz, CDCl3-d)
δ 7.37 (dd, J1 = 7.83 Hz, J2 = 1.56 Hz, 1H), 7.24 (s, 1H), 7.18 (d, J =
8.61 Hz, 1H), 7.07−7.03 (m, 3H), 3.22 (s, 3H), 2.82 (d, J = 16.42 Hz,
P
dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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1
(14 mg, 46% yield). H NMR (400 MHz, CDCl3-d) δ 8.58 (m, 1H),
fluorophenyl)-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]-
5(1H)-one to provide 2-amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-
2′H-spiro[imidazole-4,1′-naphthalene]-4′,5(1H,3′H)-dione (22 mg,
8.51 (m, 1H), 7.73 (m, 1H), 7.35−7.30 (m, 1H), 7.28 (m, 1H), 7.04
(m, 1H), 3.20 (s, 3H), 2.64 (d, J = 14.08 Hz, 1H), 2.13 (d, J = 14.08
Hz, 1H), 1.56 (s, 3H), 1.49 (s, 3H); MS (APCI+) m/z 387.2 (M + 1).
2-Amino-7′-(5-chloropyridin-3-yl)-1,3′,3′-trimethyl-3′,4′-di-
hydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (rac-
48). 2-Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro-
[imidazole-4,1′-naphthalen]-5(1H)-one (112 mg, 0.333 mmol) and
5-chloropyridin-3-ylboronic acid (68 mg, 0.433 mmol) were processed
according to the method described for the synthesis of 5-amino-6′-(5-
chloropyridin-3-yl)-1,2′,2′-trimethylspiro[pyrrole-3,4′-thiochroman]-
2(1H)-one to provide 2-amino-7′-(5-chloropyridin-3-yl)-1,3′,3′-tri-
methyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-
one (50 mg, 0.136 mmol, 41% yield). 1H NMR (400 MHz, CDCl3-d)
δ 8.60 (d, J = 2.35 Hz, 1H), 8.51 (d, J = 2.35 Hz, 1H), 7.45 (m, 1H),
7.39 (dd, J1 = 7.83 Hz, J2 = 1.56 Hz, 1H), 7.22 (d, J = 7.83 Hz, 1H),
7.06 (d, J = 1.56 Hz, 1H), 3.20 (s, 3H), 2.84 (d, J = 16.43 Hz, 1H),
2.62 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.31 (d, J = 14.09 Hz, 1H),
1.81 (dd, J1 = 14.09 Hz, J2 = 2.35 Hz, 1H), 1.17 (s, 3H), 1.02 (s, 3H).
MS (APCI+) m/z 369 (M + 1).
1
0.0475 mmol, 85% yield). H NMR (400 MHz, CD3OD-d4) δ 9.21
(s, 1H), 9.12 (s, 2H), 8.22 (d, J = 8.22 Hz, 1H), 7.97 (d, J = 8.22 Hz,
1H), 7.74 (s, 1H), 3.03 (s, 3H), 2.65 (d, J = 14.48 Hz, 1H), 2.42 (d, J
= 14.86 Hz, 1H), 1.38 (s, 3H), 1.33 (s, 3H). LCMS (APCI+) m/z
350.1 (M + 1)+, retention time 0.911 min.
2-Amino-4′,4′-difluoro-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-
3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one
(rac-52). Step A. To a solution of 2-amino-7′-bromo-1,3′,3′-trimethyl-
2′H-spiro[imidazole-4,1′-naphthalene]-4′,5(1H,3′H)-dione 2,2,2-tri-
fluoroacetate (17 mg, 0.0366 mmol) in dichloroethane (183 μL,
0.0366 mmol) at 0 °C in a plastic Falcon tube was added Deoxo-Fluor
(20.3 μL, 0.110 mmol), and the resulting mixture was stirred at 0 °C
for 15 min while warming to room temperature. The crude reaction
mixture was purified by Gilson C18 HPLC to provide TFA salt of 2-
amino-7′-bromo-4′,4′-difluoro-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-
spiro[imidazole-4,1′-naphthalen]-5(1H)-one (3 mg, 22%), which was
used for the next reaction directly.
Step B. 2-Amino-7′-bromo-4′,4′-difluoro-1,3′,3′-trimethyl-3′,4′-di-
hydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one 2,2,2-trifluor-
oacetate (3 mg, 0.00617 mmol) and pyrimidin-5-ylboronic acid (1.07
mg, 0.00864 mmol) were processed according to the method
described for the synthesis of 2-amino-6′-(3-chloro-5-fluorophenyl)-
1,2′,2′-trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one and
purified by reverse phase C-18 HPLC (5−95% gradient of CH3CN/
water) to provide 2-amino-4′,4′-difluoro-1,3′,3′-trimethyl-7′-(pyrimi-
din-5-yl)-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-
one (2.6 mg, 0.005 36 mmol, 86.8% yield). 1H NMR (400 MHz,
CD3OD-d4) δ 9.19 (s, 1H), 9.06 (s, 2H), 7.97−7.94 (m, 2H), 7.66 (m,
1H), 4.85 (s, 3H), 2.60 (d, J = 14.87 Hz, 1H), 2.31 (dd, J1 = 14.87 Hz,
J2 = 4.34 Hz, 1H), 1.29 (s, 3H), 1.21 (s, 3H); LCMS (APCI+) m/z
372.1 (M + 1)+, retention time 1.045 min.
2′-Amino-6-(3-chlorophenyl)-1′-ethyl-2,2-dimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (rac-53). Step A. To a
mixture of 6-bromo-2,2-dimethylspiro[chroman-4,4′-imidazolidine]-
2′,5′-dione (200 mg, 0.6151 mmol) and K2CO3 (850 mg, 0.6151
mmol) in DMF (4 mL) was added a solution of ethyl iodide (0.049 65
mL, 0.6151 mmol) in DMF (0.5 mL). The mixture was heated at 45
°C for 2 h and then at 60 °C for 18 h. Then an additional 0.3 equiv of
ethyl iodide and K2CO3 were added, and the mixture was heated at 60
°C for an additional 2 h. The mixture was concentrated in vacuo, and
the residue obtained was partitioned between ethyl acetate and 2 M
aqueous Na2CO3. The organic phases were combined and washed
with 2 M aqueous Na2CO3 (2×), water (1×), and brine (1×). The
organic layer was dried (Na2SO4), filtered, and concentrated in vacuo.
The residue obtained was purified on silica gel, eluting with a gradient
of 8−50% EtOAc/hexanes (10 CV) on Biotage SP1 unit to give 6-
bromo-1′-ethyl-2,2-dimethylspiro[chroman-4,4′-imidazolidine]-2′,5′-
dione (172 mg, 0.4870 mmol, 79% yield) as a pale yellow solid.
Analytical HPLC, >92 area % pure, retention time 3.808 min. FIMS
(APCI−) m/z 351/353 (M − 1) with Br isotope.
Step B. 6-Bromo-1′-ethyl-2,2-dimethylspiro[chroman-4,4′-imidazo-
lidine]-2′,5′-dione (172 mg, 0.487 mmol) and Lawesson’s reagent
(138 mg, 0.341 mmol) were processed according to the method
described for the synthesis of 6′-bromo-1,2′,2′-trimethyl-2-
thioxospiro[imidazolidine-4,4′-thiochroman]-5-one to provide 6-
bromo-1′-ethyl-2,2-dimethyl-2′-thioxospiro[chroman-4,4′-imidazoli-
din]-5′-one (108 mg, 0.292 mmol, 60% yield) as a white solid. FIMS
(APCI−) m/z 367, 369 (M − 1) with Br isotope; analytical HPLC
purity 97 area %, retention time 4.35 min.
Step C. To a solution of 6-bromo-1′-ethyl-2,2-dimethyl-2′-
thioxospiro[chroman-4,4′-imidazolidin]-5′-one (108 mg, 0.292
mmol) in MeOH (2 mL) and THF (0.7 mL) were added sequentially
tert-butyl hydroperoxide (70% aqueous) (607 μL, 4.39 mmol) and
30% NH4OH (1.14 mL, 8.77 mmol), and the reaction mixture was
heated at 40 °C. After 2 h, the mixture was concentrated under a
stream of N2, and the resulting residue was partitioned between
saturated aqueous NH4Cl and DCM (2×). The organic layers were
5-Amino-1,2′,2′-trimethyl-6′-(pyrimidin-5-yl)spiro[pyrrole-
3,4′-thiochroman]-2(1H)-one (rac-49). rac-49 was synthesized in
the same fashion as 2-amino-6′-(3-chloro-5-fluorophenyl)-1,2′,2′-
trimethylspiro[imidazole-4,4′-thiochroman]-5(1H)-one substituting
pyrimidin-5-yl boronic acid (13.4 mg, 0.108 mmol) for 3-chloro-5-
fluorophenylboronic acid to afford 5-amino-1,2′,2′-trimethyl-6′-(pyr-
imidin-5-yl)spiro[pyrrole-3,4′-thiochroman]-2(1H)-one (19 mg,
1
56.5% yield) as a tan solid. H NMR (400 MHz, DMSO-d6) δ 9.15
(s, 1H), 8.96 (s, 2H), 7.57 (m, 1H), 7.24 (m, 1H), 7.07 (m, 1H), 3.04
(s, 3H), 2.35 (m, 1H), 1.92 (m, 1H), 1.51 (s, 3H), 1.42 (s, 3H); MS
(APCI+) m/z 354.2 (M + 1); analytical HPLC retention time 2.967
min.
2-Amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-3′,4′-dihydro-
2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (rac-50). 2-
Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-
4,1′-naphthalen]-5(1H)-one (50 mg, 0.15 mmol) and pyrimidin-5-yl
boronic acid (24 mg, 0.19 mmol) were processed as described for the
synthesis of 5-amino-1,2′,2′-trimethyl-6′-(pyrimidin-5-yl)spiro-
[pyrrole-3,4′-thiochroman]-2(1H)-one to yield 2-amino-1,3′,3′-tri-
methyl-7′-(pyrimidin-5-yl)-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-
1
naphthalen]-5(1H)-one (38 mg, 0.11 mmol, 76% yield). H NMR
(400 MHz, CDCl3-d) δ 9.16 (s, 1H), 8.85 (s, 2H), 7.42 (dd, J = −7.83
Hz, J2 = 2.35 Hz, 1H), 7.09 (d, J = 1.56 Hz, 1H), 3.22 (s, 3H), 2.86 (d,
J = 15.65 Hz, 1H), 2.63 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.29 (d,
14.08 Hz, 1H), 1.84 (dd, J1 = 14.09 Hz, J2 = 2.35 Hz, 1H), 1.18 (s,
3H), 1.03 (s, 3H); 13C NMR (400 MHz, CDCl3-d) δ 179.7 (C), 157.4
(CH), 155.3 (C), 154.8 (CH), 154.8 (CH), 138.1 (C), 135.4 (C),
133.9 (C), 133.0 (C), 131.1 (CH), 126.9 (CH), 125.3 (CH), 66.0
(C), 46.9 (CH2), 43.5 (CH2), 31.2−25.9 (CH3), 29.6 (C), 26.0
(CH3); LCMS (APCI+) m/z 336.2 (M + 1)+, retention time 1.905
min (5 min run).
2-Amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-2′H-spiro-
[imidazole-4,1′-naphthalene]-4′,5(1H,3′H)-dione (rac-51). Step
A. A solution of 2-amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-
2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one (50 mg, 0.15
mmol) in acetic acid (124 μL, 0.15 mmol) was cooled to 0 °C and
treated with a solution of CrO3 (33 mg, 0.33 mmol) in acetic acid (124
μL, 0.15 mmol) and water (124 μL, 0.15 mmol). The resulting mixture
was allowed to warm to ambient temperature overnight. The reaction
mixture was diluted with water and extracted with DCM. The organic
extracts were washed with NaHCO3, brine, dried, and concentrated in
vacuo to provide crude 2-amino-7′-bromo-1,3′,3′-trimethyl-2′H-spiro-
[imidazole-4,1′-naphthalene]-4′,5(1H,3′H)-dione 2,2,2-trifluoroace-
tate (26 mg, 38% yield), which was used for the next reaction
directly. LCMS (APCI+) m/z 350, 352 (M + 1)+, retention time 1.005
min.
Step B. 2-Amino-7′-bromo-1,3′,3′-trimethyl-2′H-spiro[imidazole-
4,1′-naphthalene]-4′,5(1H,3′H)-dione 2,2,2-trifluoroacetate (26 mg,
0.056 mmol) and pyrimidin-5-yl-boronic acid (9.7 mg, 0.078 mmol)
were processed according to the synthesis of 2-amino-6′-(3-chloro-5-
Q
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combined, dried (Na2SO4), filtered, and concentrated in vacuo. The
residue obtained was purified by silica gel flash chromatography to
provide 2′-amino-6-bromo-1′-ethyl-2,2-dimethylspiro[chroman-4,4′-
imidazol]-5′(1′H)-one (41 mg, 0.116 mmol, 39.8% yield) as a white
foam. FIMS (APCI−) m/z 352/354 (M − 1) with Br isotope,
retention time 1.020 min.
2.58 (d, J = 14.09 Hz, 1H), 2.02 (d, J = 14.86 Hz, 1H), 1.53 (s, 3H),
1.43 (s, 3H); FIMS (APCI+) m/z 438 (M + 1), retention time 3.630
min.
2′-Amino-6-(3-chlorophenyl)-1′-(2-methoxyethyl)-2,2-
dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-55).
Step A. To a mixture of 6-bromo-2,2-dimethylspiro[chroman-4,4′-
imidazolidine]-2′,5′-dione (200 mg, 0.6151 mmol) and K2CO3 (85
mg, 0.6151 mmol) in DMF (4 mL) was added a solution of 1-bromo-
2-methoxyethane (86 mg, 0.615 mmol) in DMF (0.5 mL). The
mixture was heated at 45 °C for 2 h and at 60 °C overnight. The
mixture was cooled to ambient temperature and treated with an
additional 0.3 equiv of 1-bromo-2-methoxyethane and K2CO3 and
heated at 60 °C. After 20 h (total time), DMF was removed in vacuo,
and the residue obtained was partitioned between EtOAc and 2 M
aqueous Na2CO3. The organic layers were combined, washed with
water (1×), brine (1×), dried (Na2SO4), filtered, and concentrated in
vacuo. The residue obtained was purified on silica gel, eluting with a
gradient of 8−66% EtOAc/hexanes (10 CV) followed by 66% EtOAc/
hexanes (2 CV) to give 6-bromo-1′-(2-methoxyethyl)-2,2-
dimethylspiro[chroman-4,4′-imidazolidine]-2′,5′-dione (196 mg,
0.511 mmol, 83% yield) as a yellow solid. FIMS (APCI−) m/z
381/383 (M − 1) with Br isotope; HPLC purity >90 area%, retention
time 3.702 min.
Step B. 6-Bromo-1′-(2-methoxyethyl)-2,2-dimethylspiro[chroman-
4,4′-imidazolidine]-2′,5′-dione (196 mg, 0.511 mmol) and Lawesson’s
reagent (145 mg, 0.358 mmol) were processed according to the
method described for the synthesis of 6′-bromo-1,2′,2′-trimethyl-2-
thioxospiro[imidazolidine-4,4′-thiochroman]-5-one to provide 6-
bromo-1′-(2-methoxyethyl)-2,2-dimethyl-2′-thioxospiro[chroman-
4,4′-imidazolidin]-5′-one (104 mg, 0.260 mmol, 51% yield) as a white
solid. FIMS (APCI−) m/z 397/399 (M − 1) with Br isotope; HPLC
purity 96 area %, retention time 4.211 min.
Step C. 6-Bromo-1′-(2-methoxyethyl)-2,2-dimethyl-2′-thioxospiro-
[chroman-4,4′-imidazolidin]-5′-one (104 mg, 0.260 mmol), tert-butyl
hydroperoxide (70% aqueous) (0.541 mL, 3.91 mmol), and 30%
NH4OH (1.01 mL, 7.81 mmol) were processed according to the
method described for the synthesis of 2′-amino-6-bromo-2,2-dimethyl-
1′-(2,2,2-trifluoroethyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one to
provide 2′-amino-6-bromo-1′-(2-methoxyethyl)-2,2-dimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (90 mg, 0.235 mmol, 90%
yield) as a white foam. FIMS (APCI−) m/z 382/384 (M − 1) with
Br isotope.
Step D. 2′-Amino-6-bromo-1′-(2-methoxyethyl)-2,2-dimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (90 mg, 0.235 mmol) and 3-
chlorophenylboronic acid (44 mg, 0.283 mmol) were processed
according to the method described for the synthesis of 2-amino-6′-(3-
chloro-5-fluorophenyl)-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochro-
man]-5(1H)-one to provide 2′-amino-6-(3-chlorophenyl)-1′-(2-me-
thoxyethyl)-2,2-dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one
(23 mg, 0.0556 mmol, 24% yield) as a yellow powder. 1H NMR
(CDCl3-d) δ 7.40 (s, 1H), 7.36 (dd, J1 = 8.61 Hz, J2 = 2.35 Hz, 1H),
7.30−7.25 (m, 2H), 7.24−7.21 (m, 1H), 6,92 (d, J = 8.61 Hz, 1H),
6.88 (s, 1H), 3.94−3.85 (m, 1H), 3.80−3.72 (m, 1H), 3.64−3.54 (m,
2H), 3.42 (s, 3H), 2.49 (d, J = 14.09 Hz, 1H), 1.94 (d, J = 14.09 Hz,
1H), 1.52 (s, 3H), 1.47 (s, 3H); FIMS (APCI+) m/z 414 (M + 1),
retention time 3.480 min.
Step D. The compound was synthesized in the same fashion as 2-
amino-6′-(3-chloro-5-fluorophenyl)-1,2′,2′-trimethylspiro[imidazole-
4,4′-thiochroman]-5(1H)-one, substituting 3-chlorophenylboronic
acid (22 mg, 0.140 mmol) for 3-chloro-5-fluorophenylboronic acid
to provide 2′-amino-6-(3-chlorophenyl)-1′-ethyl-2,2-dimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (17 mg, 0.0443 mmol, 38%
1
yield) as a white powder. H NMR (CDCl3-d) δ 7.44−7.35 (m, 2H),
7.33−7.27 (m, 3H), 6.70−6.91 (m, 2H), 3.75−3.67 (m, 2H), 2.56 (d, J
= 14.09 Hz, 1H), 1.98 (d, J = 14.87 Hz, 1H), 1.51 (s, 3H), 1.41 (s,
3H), 1.33 (t, J = 7.04 Hz, 3H); FIMS (APCI+) m/z 384 (M + 1);
analytical HPLC purity >99 area %, retention time 3.415 min.
2′-Amino-6-(3-chlorophenyl)-2,2-dimethyl-1′-(2,2,2-
trifluoroethyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (rac-
54). Step A. To a mixture of 6-bromo-2,2-dimethylspiro[chroman-
4,4′-imidazolidine]-2′,5′-dione (200 mg, 0.615 mmol) and K2CO3
(850 mg, 0.615 mmol) in DMF (1.5 mL) at ambient temperature was
added a solution of 1,1,1-trifluoro-2-iodoethane (129 mg, 0.615 mmol)
in DMF (1 mL). The reaction mixture was heated at 60 °C for 18 h
and then at 85 °C and stirred for another 5 days. The mixture was
cooled to ambient temperature and partitioned between saturated
NH4Cl solution and DCM (2×). The combined organic layers were
dried (Na2SO4), filtered, and concentrated in vacuo. The crude was
purified on silica gel (Snap 50 cartridge), eluting with a gradient of
10−50% EtOAc/hexane (8 CV), followed by 50% EtOAc/hexanes (2
CV) to give 6-bromo-2,2-dimethyl-1′-(2,2,2-trifluoroethyl)spiro-
[chroman-4,4′-imidazolidine]-2′,5′-dione (155 mg, 0.381 mmol, 62%
yield) as a white foam. FIMS (APCI−) m/z 405/407 (M − 1) with Br
isotope, retention time 4.054 min
Step B. 6-Bromo-2,2-dimethyl-1′-(2,2,2-trifluoroethyl)spiro-
[chroman-4,4′-imidazolidine]-2′,5′-dione (155 mg, 0.381 mmol) and
Lawesson’s reagent (108 mg, 0.266 mmol) were processed according
to the procedure described for the synthesis of 6′-bromo-1,2′,2′-
trimethyl-2-thioxospiro[imidazolidine-4,4′-thiochroman]-5-one to give
6-bromo-2,2-dimethyl-2′-thioxo-1′-(2,2,2-trifluoroethyl)spiro-
[chroman-4,4′-imidazolidin]-5′-one (50 mg, 0.118 mmol, 31% yield)
as a clear colorless residue. LC/MS (ESI−) m/z 421/423 (M − 1)
with Br isotope; analytical HPLC purity 96 area %, retention time
4.379 min.
Step C. To a mixture of 6-bromo-2,2-dimethyl-2′-thioxo-1′-(2,2,2-
trifluoroethyl)spiro[chroman-4,4′-imidazolidin]-5′-one (50 mg, 0.118
mmol) in MeOH (0.6 mL) and THF (0.15 mL) was added
sequentially 70% aqueous tert-butyl hydroperoxide (0.245 mL, 1.77
mmol) and 30% NH4OH (0.460 mL, 3.54 mmol), and the reaction
mixture was heated at 40 °C with stirring. After 2 h, the reaction
mixture was diluted with water (2 mL), and the organics were
removed in vacuo. The resulting mixture was partitioned between
saturated aqueous NH4Cl and DCM (2×). The combined organic
extracts were dried (Na2SO4), filtered, and concentrated in vacuo. The
crude was purified on silica gel (Snap 10g), eluting with a gradient of
1−8% MeOH/DCM (15 CV) followed by 8% MeOH/DCM (5 CV)
on Biotage SP1 unit to provide 2′-amino-6-bromo-2,2-dimethyl-1′-
(2,2,2-trifluoroethyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one (12
mg, 25% yield) as a white solid. LC/MS (APCI+) m/z 406/408 (M
+ 1) with Br isotope.
1′-((1-Acetylpiperidin-4-yl)methyl)-2′-amino-6-(3-chloro-
phenyl)-2,2-dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one
(rac-56). Step A. To a solution of 2′-amino-6-bromo-2,2-
dimethylspiro[chroman-4,4′-imidazol]-5′(1′H)-one (100 mg, 0.308
mmol) in DMF (1.2 mL) was added DMF dimethylacetal (0.131 mL,
0.925 mmol). The reaction mixture was stirred at ambient temperature
for 18 h and concentrated in vacuo. The resulting residue was
dissolved in minimal DCM, and this solution was added dropwise to
vigorously stirring hexanes, causing precipitation. The solids were
isolated by vacuum filtration (0.2 μm nylon filter membrane), rinsed
with hexanes, air-dried, and dried in vacuo to give (E)-N′-(6-bromo-
2,2-dimethyl-5′-oxo-1′,5′-dihydrospiro[chroman-4,4′-imidazole]-2′-
yl)-N,N-dimethylformimidamide (80 mg, 0.211 mmol, 68.4% yield) as
Step D. 2′-Amino-6-bromo-2,2-dimethyl-1′-(2,2,2-trifluoroethyl)-
spiro[chroman-4,4′-imidazol]-5′(1′H)-one (12 mg, 0.0295 mmol)
and 3-chlorophenylboronic acid (7 mg, 0.0443 mmol) were processed
according to the method described for the synthesis of 2-amino-6′-(3-
chloro-5-fluorophenyl)-1,2′,2′-trimethylspiro[imidazole-4,4′-thiochro-
man]-5(1H)-one to provide 2′-amino-6-(3-chlorophenyl)-2,2-dimeth-
yl-1′-(2,2,2-trifluoroethyl)spiro[chroman-4,4′-imidazol]-5′(1′H)-one
1
(11 mg, 0.0251 mmol, 85% yield) as a white solid. H NMR (CDCl3-
d) δ 7.68−7.63 (m, 1H), 7.48−7.46 (m, 1H), 7.43−7.40 (m, 2H),
7.31−7.29 (m, 2H), 6.94 (d, J = 8.61 Hz, 1H), 4.40−4.19 (m, 2H),
1
a white powder. H NMR (DMSO-d6) δ 9.39 (s, 1H),8.78 (s, 1H),
R
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Journal of Medicinal Chemistry
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7.31 (dd, J1 = 9.39 Hz, J2 = 2.35 Hz, 1H), 6.80 (d, J = 2.35 Hz, 1H),
6.75 (d, J = 8.61 Hz, 1H), 3.22 (s, 3H), 3.10 (s, 3H), 2.27 (d, J = 14.09
Hz, 1H), 1.94 (d, J = 14.09 Hz, 1H),1.41 (s, 3H), 1.27 (s, 3H); LC/
MS (APCI+) m/z 379/381 (M + 1).
4,4′-imidazol]-5′(1′H)-one (11.6 mg, 0.0234 mmol, 77% yield) as a
white residue. LC/MS (APCI+) m/z 495 (M + 1), retention time
1.024 min.
7-(5-Chloropyridin-3-yl)-3,3-dimethyl-3,4-dihydro-2H,5′H-
spiro[naphthalene-1,4′-oxazol]-2′-amine (rac-57). rac-57 was
synthesized in the same fashion as compound 62 (below), replacing
2-fluoropyridin-3-ylboronic acid with (5-chloropyridin-3-yl)boronic
acid to afford 7-(5-chloropyridin-3-yl)-3,3-dimethyl-3,4-dihydro-
2H,5′H-spiro[naphthalene-1,4′-oxazol]-2′-amine (8 mg, 0.023 mmol,
Step B. To a mixture of (E)-N′-(6-bromo-2,2-dimethyl-5′-oxo-1′,5′-
dihydrospiro[chroman-4,4′-imidazole]-2′-yl)-N,N-dimethylformimida-
mide (47 mg, 0.124 mmol) and K2CO3 (26 g, 0.186 mmol) in DMF
(0.6 mL) was added tert-butyl 4-(bromomethyl)piperidine-1-carbox-
ylate (41.4 mg, 0.149 mmol). The mixture was heated to 60 °C and
stirred overnight. After 16 h, additional K2CO3 (26 mg, 0.186 mmol)
and tert-butyl 4-(bromomethyl)piperidine-1-carboxylate (42 mg, 0.149
mmol) were added, and the reaction mixture was heated to 80 °C for 5
h and then at 90 °C for 2 more days. The reaction mixture was cooled
to ambient temperature and concentrated under a nitrogen stream.
The resulting residue was suspended in DCM, sonicated, and the
resulting solids were removed by filtration. The filtrate collected was
concentrated in vacuo, and the isolated crude was purified by
preparative TLC, eluting with 9:1 DCM/MeOH to give (E)-tert-butyl
4-((6-bromo-2′-((dimethylamino)methyleneamino)-2,2-dimethyl-5′-
oxospiro[chroman-4,4′-imidazole]-1′(5′H)-yl)methyl)piperidine-1-
carboxylate (50 mg, 0.0867 mmol, 70% yield) as a clear colorless
residue. LCMS (APCI+) m/z 576/578 (M + 1) with Br isotope; LC/
MS purity 96 area %, retention time 1.347 min.
Step C. To a solution of (E)-tert-butyl 4-((6-bromo-2′-
((dimethylamino)methyleneamino)-2,2-dimethyl-5′-oxospiro-
[chroman-4,4′-imidazole]-1′(5′H)-yl)methyl)piperidine-1-carboxylate
(43 mg, 0.0746 mmol) in DCM (0.7 mL) at ambient temperature was
added neat TFA (0.115 mL, 1.49 mmol), and the mixture was stirred
for 1 h. The mixture was then concentrated under a nitrogen stream to
provide the crude (E)-N′-(6-bromo-2,2-dimethyl-5′-oxo-1′-(piperidin-
4-ylmethyl)-1′,5′-dihydrospiro[chroman-4,4′-imidazole]-2′-yl)-N,N-di-
methylformimidamide (43 mg, 0.0750 mmol, 101% yield), which was
used for the next reaction directly.
Step D. To a solution of crude (E)-N′-(6-bromo-2,2-dimethyl-5′-
oxo-1′-(piperidin-4-ylmethyl)-1′,5′-dihydrospiro[chroman-4,4′-imida-
zole]-2′-yl)-N,N-dimethylformimidamide (53 mg, 0.0924 mmol) and
TEA (0.0515 mL, 0.370 mmol) in DCM (0.8 mL) at ambient
temperature was added acetyl chloride (0.00923 mL, 0.129 mmol),
and the mixture was stirred for 2 h. The reaction mixture was
concentrated under a nitrogen stream, and the crude was purified by
preparative TLC (1 mm plate, 19:1 DCM/MeOH) to give (E)-N′-(1′-
((1-acetylpiperidin-4-yl)methyl)-6-bromo-2,2-dimethyl-5′-oxo-1′,5′-
dihydrospiro[chroman-4,4′-imidazole]-2′-yl)-N,N-dimethylformimida-
mide (23 mg, 0.0444 mmol, 48.0% yield) as a white residue. LCMS
(APCI+) m/z 518/520 (M + 1) with Br isotope.
Step E. A sealable glass tube was charged with a mixture of (E)-N′-
(1′-((1-acetylpiperidin-4-yl)methyl)-6-bromo-2,2-dimethyl-5′-oxo-
1′,5′-dihydrospiro[chroman-4,4′-imidazole]-2′-yl)-N,N-dimethylformi-
midamide (18 mg, 0.034 72 mmol) and 7 N NH3 in MeOH (0.4960
mL, 3.472 mmol). The tube was capped and heated at 50 °C. After 3
days, the mixture was cooled to ambient temperature and treated with
additional 7 N NH3 in MeOH (100 equiv) and heated at 60 °C with
stirring. After 28 h, the mixture was cooled to ambient temperature,
concentrated in vacuo, and the residue obtained was purified by
preparative TLC (1 mm, 9:1 DCM/7 N NH3/MeOH) to give 1′-((1-
acetylpiperidin-4-yl)methyl)-2′-amino-6-bromo-2,2-dimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (14 mg, 0.03021 mmol, 87%
yield) as a white residue. LCMS (APCI+) m/z 463/465 (M + 1) with
Br isotope.
1
29% yield). H NMR (400 MHz, CDCl3-d) δ 8.69 (d, J = 2 Hz, 1H),
8.51 (d, J = 2 Hz, 1H), 7.82 (m, 1H), 7.44 (d, J = 2 Hz, 1H), 7.35 (dd,
J1 = 7.8 Hz, J2 = 2 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 4.35 (dd, J1 = 8.2
Hz, J2 = 4.3 Hz, 2H), 2.7 (d, 16 Hz, 1H), 2.57 (dd, J1 = 16 Hz, J2 = 1.2
Hz, 1H), 2.06 (d, J = 14 Hz, 1H), 1.86 (dd, J1 = 14 Hz, J2 = 1.6 Hz,
1H), 1.1 (s, 3H), 0.98 (s, 3H); LCMS (APCI+) m/z 342.1 (M + 1).
7-(5-Chloropyridin-3-yl)-3,3-dimethyl-3,4-dihydro-2H,5′H-
spiro[naphthalene-1,4′-thiazol]-2′-amine (rac-58). rac-58 was
synthesized in the same fashion as compound 57, substituting silver
cyanate with silver thiocyanate to afford 7-(5-chloropyridin-3-yl)-3,3-
dimethyl-3,4-dihydro-2H,5′H-spiro[naphthalene-1,4′-thiazol]-2′-
amine (10 mg, 0.028 mmol, 45% yield). 1H NMR (400 MHz, CDCl3-
d) δ 8.69 (d, J = 2 Hz, 1H), 8.51 (d, J = 2 Hz, 1H), 7.84 (t, J = 2 Hz,
1H), 7.63 (d, J = 2 Hz, 1H), 7.35 (dd, J1 = 7.8 Hz, J2 = 2 Hz, 1H), 7.18
(d, J = 7.8 Hz, 1H), 3.65 (dd, J1 = 11 Hz, J2 = 10 Hz, 2H), 2.7 (d, 16
Hz, 1H), 2.6 (dd, J1 = 16 Hz, J2 = 1.2 Hz, 1H), 2.07 (dd, J1 = 14 Hz, J2
= 1.6 1H), 1.9 (d, J1 = 14 Hz, 1H), 1.09 (s, 3H), 1.07 (s, 3H); LCMS
(APCI+) m/z 358.1 (M + 1).
7-(5-Chloropyridin-3-yl)-3,3-dimethyl-3,4,5′,6′-tetrahydro-
2H-spiro[naphthalene-1,4′-[1,3]thiazin]-2′-amine (rac-59). Step
A. A solution of vinyl magnesium bromide 1 M in THF (39.5 mL, 39.5
mmol) was added dropwise to a solution of 7-bromo-3,3-dimethyl-3,4-
dihydronaphthalen-1(2H)-one (5 g, 19.8 mmol) in THF at 0 °C under
N2. After 2 h, the reaction mixture was poured into ice cold saturated
NH4Cl solution (150 mL) and extracted into EtOAc (2 × 100 mL).
The combined organic layers were washed with brine, then dried
(MgSO4) and concentrated in vacuo to provide the crude 7-bromo-
3,3-dimethyl-1-vinyl-1,2,3,4-tetrahydronaphthalen-1-ol as a liquid (5.7
1
g, 83% yield). H NMR (400 MHz, CDCl3-d) δ 7.40 (d, J = 1.96 Hz,
1H), 7.33 (dd, J1 = 8.22 Hz, J2 = 2.35 Hz, 1H), 7.04 (d, J = 8.22 Hz,
1H), 5.91 (dd, J1 = 16.82 Hz, J2 = 10.17 Hz, 1H), 5.27 (dd, J1 = 16.84
Hz, J2 = 1.96 Hz, 1H), 5.06 (dd, J1 = 10.17 Hz, J2 = 1.96 Hz, 1H), 5.03
(s, 1H, exchanged with D2O), 2.45 (m, 2H), 1.74 (d, J = 14.06 Hz,
1H), 1.68 (d, J = 14.06 Hz, 1H), 0.98 (s, 3H), 0.96 (s, 3H).
Step B. To a solution of crude 7-bromo-3,3-dimethyl-1-vinyl-
1,2,3,4-tetrahydronaphthalen-1-ol (5.55g, 19.7 mmol) in CH3CN (50
mL) at 0 °C was added thionyl chloride (2.88 mL, 39.5 mmol). The
reaction mixture was stirred at 0 °C for 10 min and treated with
thiourea (3.0 g, 39.5 mmol) in one portion. The mixture was then
allowed to stir at ambient temperature for 10 min and then at 50 °C
for 1 h. The resulting mixture was allowed to stir at ambient
temperature over the weekend. The solid formed was filtered and
washed with additional CH3CN (3 × 5 mL) and dried to provide (E)-
2-(7-bromo-3,3-dimethyl-3,4-dihydronaphthalen-1(2H)-ylidene)ethyl
1
carbamimidothioate hydrochloride (7.9 g, 90.5% yield) as a solid. H
NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 2.35 Hz, 1H), 7.37 (dd, J1
= 8.22 Hz, J2 = 1.96 Hz, 1H), 7.09 (d, J = 8.22 Hz, 1H), 6.31 (t, J =
8.22 Hz, 1H), 4.10 (d, J = 7.83 Hz, 2H), 2.58 (s, 2H), 2.3 (s, 2H), 0.93
(s, 6H); LCMS (APCI+) m/z 400 (M + 1)+; analytical HPLC purity
91 area % (254 nM), retention time 3.276 min.
Step C. A solution of (E)-2-(7-bromo-3,3-dimethyl-3,4-dihydro-
naphthalen-1(2H)-ylidene)ethyl carbamimidothioate hydrochloride
(7.8 g, 21 mmol) in 2,2,2-trifluoroacetic acid (21 mL, 21 mmol) at
0 °C was treated dropwise with methanesulfonic acid (10 mL, 21
mmol). The resulting mixture was stirred at 0 °C for 30 min and then
at ambient temperature overnight. The mixture was then cooled to 0
°C and slowly poured into ice cold saturated Na2CO3 solution (200
mL). The resulting slurry was stirred at ambient temperature for 30
min. The solid formed was filtered, washed with copious amount of
water, then triturated with hot MeOH and filtered. The filtrate
collected was concentrated in vacuo and dried to provide 7-bromo-3,3-
Step F. A sealable glass vial was charged with a mixture of 1′-((1-
acetylpiperidin-4-yl)methyl)-2′-amino-6-bromo-2,2-dimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one (14 mg, 0.0302 mmol), 3-
chlorophenylboronic acid (6.14 mg, 0.0393 mmol),
PdCl2(dppf)·DCM (2.47 mg, 0.003 02 mmol), dioxane (0.3 mL),
and 2 M aqueous Na2CO3 (0.0604 mL, 0.121 mmol). The mixture was
purged with nitrogen, vial capped, and the reaction mixture was heated
at 90 °C and stirred for 90 min. The reaction mixture was diluted with
DCM (0.2 mL) and purified by preparative TLC plate (1 mm plate,
9:1 DCM/7 N NH3/MeOH) to give 1′-((1-acetylpiperidin-4-yl)-
methyl)-2′-amino-6-(3-chlorophenyl)-2,2-dimethylspiro[chroman-
S
dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
dimethyl-3,4,5′,6′-tetrahydro-2H-spiro[naphthalene-1,4′-[1,3]thiazin]-
2′-amine (5.2 g, 74% yield) as a solid. 1H NMR (DMSO-d6) δ 9.99 (br
s, 1H), 7.70 (s, 1H), 7.50 (dd, J1 = 8.22 Hz, J2 = 1.96 Hz, 1H), 7.16 (d,
J = 7.83 Hz, 1H), 3.39 (td, J1 = 12.91 Hz, J2 = 3.91 Hz, 1H), 3.25 (d,t,
J1 = 12.91 Hz, J2 = 3.91 Hz, 1H), 2.55 (d, J = 5.87 Hz, 2H), 2.29−2.30
(m, 1H), 2.29−2.14 (m, 1H), 1.85 (s, 2H), 1.05 (s, 3H), 0.87 (s, 3H);
LCMS (APCI+) m/z 340 (M + 1); analytical HPLC purity 96 area %,
retention time 3.032 min.
internal temperature below −74 °C. The resulting mixture was stirred
at −78 °C for 1 h and treated dropwise with a solution of (E)-N-(7-
bromo-3,3-dimethyl-3,4-dihydronaphthalen-1(2H)-ylidene)-2-methyl-
propane-2-sulfinamide (4.15 g, 11.6 mmol) in THF (70 mL) at a rate
such that the internal temperature did not excess −74 °C. Once the
addition was complete, the mixture was stirred at −78 °C for 3 h. The
mixture was then poured into a saturated NaHCO3 solution (100 mL)
and extracted into EtOAc (3 × 50 mL). The organic layers were
combined, dried (MgSO4), and concentrated in vacuo. The crude
isolated was purified by flash chromatography on silica gel (Ready Sep
220 g), eluting with a step gradient of 20% EtOAc/hexane (to recover
unreacted SM) and then with 30%EtOAc/hexane followed by 50%
EtOAc/hexane to provide methyl 2-((1R)-7-bromo-1-(1,1-dimethyle-
thylsulfinamido)-3,3-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)-
Step D. A resealable glass pressure tube was charged with 7-bromo-
3,3-dimethyl-3,4,5′,6′-tetrahydro-2H-spiro[naphthalene-1,4′-[1,3]-
thiazin]-2′-amine (40 mg, 0.12 mmol), 5-chloropyridin-3-ylboronic
acid (22 mg, 0.14 mmol), Pd(dppf)·DCM complex (4.7 mg, 0.0059
mmol), 20% aqueous Na2CO3 (219 μL, 0.41 mmol), and 1,4-dioxane
(1179 μL, 0.12 mmol). The mixture was sparged with N2 for 5 min,
capped, and stirred at 90 °C for 15 h, and allowed to cool temperature.
The mixture was diluted with EtOAc (10 mL) and washed with brine
(2 × 5 mL). The organic layer was separated, dried (MgSO4), and
concentrated in vacuo. The residue obtained was passed through a
silica plug (2 g), eluting with 10% MeOH/DCM + 1% NH3. The
semipure material obtained was purified by C-18 reverse phase HPLC
(Gilson Unipoint), eluting with a gradient of 5−95% CH3CN/water
containing 0.1% TFA to provide the TFA salt of 7-(5-chloropyridin-3-
yl)-3,3-dimethyl-3,4,5′,6′-tetrahydro-2H-spiro[naphthalene-1,4′-[1,3]-
1
acetate as a white solid (2.28 g, 45% yield). H NMR (400 MHz,
DMSO-d6) δ 7.65 (d, J = 2.35 Hz, 1H), 7.36 (dd, J1 = 7.82 Hz, J2 =
1.96 Hz, 1H), 7.06 (d, J = 8.22 Hz, 1H), 5.36 (s, 1H), 3.60 (s, 3H),
3.05 (d, J = 15.65 Hz, 1H), 2.86 (d, J = 16.04 Hz, 1H), 2.54 (d, J =
15.65 Hz, 1H), 2.48−2.46 (m, 1H), 2.36-d, J = 14.87 Hz, 1H), 1.94 (d,
J = 14.87 Hz, 1H), 1.12 (s, 9H), 0.98 (s, 3H), 0.92 (s, 3); FIMS (APCI
+) m/z 429.8, 431.8; analytical HPLC purity 100 area %, retention
time 4.473 min.
Step C. A solution of methyl 2-(7-bromo-1-(1,1-dimethylethylsulfi-
namido)-3,3-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)acetate (2.27
g, 5.27 mmol) in dioxane (10 mL) was treated with 4 M HCl in
dioxane (6.59 mL, 26.4 mmol). The resulting solution was stirred at
room temperature for 2.5 h. The solvent was removed in vacuo, and
the residue was evaporated from DCM. The residue obtained was
dried under high vacuum for 30 min to provide methyl 2-(1-amino-7-
bromo-3,3-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)acetate (1.7 g,
1
thiazin]-2′-amine (8 mg, 0.022 mmol, 18% yield) as a white solid. H
NMR (400 MHz, CDCl3-d) δ 11.66 (s, 1H), 6.30 (m, 2H), 7.92 (s,
1H), 7.44 (m, 2H), 7.28 (m, 1H), 3.37−3.30 (m, 1H), 3.13−3.01 (m,
1H), 2.37−2.30 (m, 1H), 2.23−2.14 (m, 3H), 2.12 (d, J = 13.69 Hz,
1H), 1.85 (d, J = 14.09 Hz, 1H), 1.17 (s, 3H), 1.01 (s, 3H); LCMS
(APCI+) m/z 372 (M + 1), retention time 2.334 min.
2′-Amino-7-(5-chloropyridin-3-yl)-1′,3,3-trimethyl-3,4-dihy-
dro-1′H,2H-spiro[naphthalene-1,4′-pyrimidin]-6′(5′H)-one
(rac-60). Step A. To a solution of 7-bromo-3,3-dimethyl-3,4-
dihydronaphthalen-1(2H)-one (5 g, 19.8 mmol) and 2-methylpro-
pane-2-sulfinamide (3.11 g, 25.7 mmol) in THF (65.8 mL, 19.8
mmol) was added freshly distilled Ti(OEt)4 (9.01 g, 39.5 mmol) in
one portion. The resulting mixture was refluxed for 18 h. The mixture
was cooled to ambient temperature and poured into saturated
NaHCO3 (500 mL) solution. The resulting suspension was shaken
with EtOAc (300 mL) and filtered through a pad of Celite 545. The
solid particles in the filter funnel were crushed with a spatula and
washed well with EtOAc (∼200 mL) until all the yellow color
disappeared from the solids. The filtrate was transferred to a separatery
funnel, and the layers were separated. The aqueous layer was extracted
once with EtOAc (100 mL). The combined organic layers were
washed with brine (1 × 100 mL), dried (MgSO4), filtered, and
concentrated in vacuo. The residue obtained was purified by flash
chromatography on silica gel (Ready Sep 120 g), eluting with 15%
EtOAc (2 L) followed by 20% EtOAc/hexane (500 mL) to provide
(E)-N-(7-bromo-3,3-dimethyl-3,4-dihydronaphthalen-1(2H)-ylidene)-
2-methylpropane-2-sulfinamide (4.2 g, 11.8 mmol, 59.7% yield) as a
yellow solid. The unreacted SM 7-bromo-3,3-dimethyl-3,4-dihydro-
naphthalen-1(2H)-one was also recovered from this reaction. 1H
NMR (400 MHz, CDCl3-d) δ 8.22 (d, J = 1.96 Hz, 1H), 7.50 (dd, J1 =
8.88 Hz, J2 = 2.35 Hz, 1H), 7.04 (d, J = 8.22 Hz, 1H), 3.14 (d, J =
16.82 Hz, 1H), 2.87 (dd, J1 = 16.82 Hz, J2 = 0.783 Hz, 1H), 2.72 (d, J
= 16.43 Hz, 1H), 2.61 (d, J = 16.43 Hz, 1H), 1.33 (s, 9H), 1.07 (s,
3H), 1.01 (s, 3H); LCMS (APCI+) m/z 356, 359 (M + 1); analytical
HPLC purity 97 area %, retention time 4.691 min.
Step B. A round-bottom flask equipped with a N2 inlet, rubber
septum, and an internal temperature probe was charged with a
solution of diisopropylamine (3.44 mL, 24.5 mmol) in THF (25 mL).
The solution was cooled to −78 °C and treated dropwise with n-
butyllithium 2.5 M in hexanes (9.78 mL, 24.5 mmol). Once the
addition was complete, the cooling bath was replaced with an ice bath,
and the mixture was allowed to stir at 0 °C for 40 min. Meanwhile a
separate round-bottom flask equipped with a N2 inlet, rubber septum,
and an internal temperature probe was charged with a solution of
methyl acetate (2.04 mL, 25.6 mmol) in THF (20 mL). The mixture
was cooled to −78 °C. Then the above-prepared LDA solution in
THF was slowly added to this solution via a cannula, maintaining
1
5.21 mmol, 98.8% yield) as a pale yellow gum. H NMR (400 MHz,
DMSO-d6) δ 7.75 (d, J = 1.96 Hz, 1H), 7.31 (dd, J1 = 1.96 Hz, J2 =
7.83 Hz, 1H), 7.02 (d, J = 8.22 Hz, 1H), 3.51 (s, 3H), 2.79 (d, J =
15.26 Hz, 1H), 2.71 (d, J = 14.87 Hz, 1H), 2.45−2.41 (m, 2H), 1.97
(d, J = 14.06 Hz, 1H), 1.63 (dd, J1 = 14.06 Hz, J2 = 1.57 Hz, 1H), 0.99
(s, 3H), 0.89 (s, 3H); LCMS (APCI+) m/z 325.8, 327.7 (M + 1).
Step D. A suspension of methyl 2-(1-amino-7-bromo-3,3-dimethyl-
1,2,3,4-tetrahydronaphthalen-1-yl)acetate (500 mg, 1.53 mmol), EDCI
(529 mg, 2.76 mmol), and methyl carbamothioylcarbamate (10) (437
mg, 2.30 mmol) in N,N-dimethylformamide (7663 μL, 1.53 mmol)
was treated with N-ethyl-N-isopropylpropan-2-amine (1303 μL, 7.66
mmol) and stirred at ambient temperature for 18 h. The mixture was
poured into water (50 mL) and extracted with EtOAc (3 × 40 mL).
The organic layers were combined, dried (MgSO4), and concentrated
in vacuo. The residue obtained was crystallized from MeOH to
provide tert-butyl 7-bromo-1′,3,3-trimethyl-6′-oxo-3,4,5′,6′-tetrahydro-
1′H,2H-spiro[naphthalene-1,4′-pyrimidine]-2′-ylcarbamate (480 mg,
1
69.5% yield) as a solid. H NMR (400 MHz, DMSO-d6) δ 9.962 (s,
1H), 7.91 (d, J = 1.96 Hz, 1H), 7.50 (dd, J1 = 8.22 Hz, J2 = 1.96 Hz,
1H), 7.15 (d, J = 8.22 Hz, 1H), 3.48 (d, J = 15.65 Hz, 1H), 3.16 (s,
3H), 2.11 (dd, J1 = 15.65 Hz, J2 = 1.56 Hz, 1H), 1.92 (d, J = 14.08 Hz,
1H), 1.76 (d, J = 14.09 Hz, 1H), 1.36 (s, 2H), 0.99 (s, 3H), 0.87 (s,
3H); LCMS (APCI+) m/z 450, 451 (M + 1) with bromine isotope.
Step E. tert-Butyl 7-bromo-1′,3,3-trimethyl-6′-oxo-3,4,5′,6′-tetrahy-
dro-1′H,2H-spiro[naphthalene-1,4′-pyrimidine]-2′-ylcarbamate (63
mg, 0.14 mmol), 5-chloropyridin-3-ylboronic acid (26 mg, 0.17
mmol), dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II)
dichloromethane adduct (5.8 mg, 0.0070 mmol), 20% aqueous
Na2CO3 (259 μL, 0.49 mmol), and 1,4-dioxane (1399 μL, 0.14
mmol) were processed according to the method described for the
synthesis of 7-(5-chloropyridin-3-yl)-3,3-dimethyl-3,4,5′,6′-tetrahydro-
2H-spiro[naphthalene-1,4′-[1,3]thiazin]-2′-amine to provide depro-
tected 2′-amino-7-(5-chloropyridin-3-yl)-1′,3,3-trimethyl-3,4-dihydro-
1′H,2H-spiro[naphthalene-1,4′-pyrimidin]-6′(5′H)-one (42 mg, 0.11
mmol, 78% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ
9.76 (s, 1H), 8.95 (d, J = 1.96 Hz, 1H), 8.64 (d, J = 2.34 Hz, 1H), 8.33
(t, J = 1.96 Hz, 1H), 8.16 (d, J = 1.96 Hz, 1H), 7.80 (dd, J1 = 8.22 Hz,
J2 = 1.96 Hz, 1H), 7.33 (d, J = 7.83 Hz, 1H), 3.77 (d, J = 16.04 Hz,
1H), 3.28 (s, 3H), 2.81 (d, J = 16.82 Hz, 1H), 2.67 (d, J = 16.04 Hz,
1H), 2.60 (d, J = 16.44 Hz, 1H), 2.04 (dd, J1 = 14.09 Hz, J2 = 1.57 Hz,
T
dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1H), 1.82 (d, J = 14.09 Hz, 1H), 1.03 (s, 3H), 0.94 (s, 3H); LCMS
(APCI+) m/z 283 (M + 1).
Hz, 2H), 2.7 (d, 16 Hz, 1H), 2.57 (dd, J1 = 16 Hz, J2 = 1.2 Hz, 1H),
2.06 (d, J = 14 Hz, 1H), 1.86 (dd, J1 = 14 Hz, J2 = 1.6 Hz, 1H), 1.1 (s,
3H), 0.98 (s, 3H); LCMS (APCI+) m/z 326.1 (M + 1).
7-(2-Fluoropyridin-3-yl)-3,3-dimethyl-3,4-dihydro-2H,5′H-
spiro[naphthalene-1,4′-oxazol]-2′-amine (rac-62). Step A. NaH
(94.8 mg, 3.95 mmol) was added to DMSO (6 mL) in a round-bottom
flask. The mixture was heated to 75 °C and stirred for 30 min. The
mixture was cooled to 0 °C, and methyltriphenylphosphonium
bromide (1411 mg, 3.95 mmol) was added dropwise in warm
DMSO (4 mL). After the mixture was stirred for 15 min, 7-bromo-3,3-
dimethyl-3,4-dihydronaphthalen-1(2H)-one (500 mg, 1.98 mmol) in
DMSO (4 mL) was added dropwise. The mixture was allowed to
warm to ambient temperature and stirred overnight. The mixture was
diluted with ethyl acetate and water. The layers were separated, and
the organics were washed with water and brine, dried over MgSO4,
filtered, and concentrated. The material was purified on silica gel.
Elution with hexanes to yield 7-bromo-3,3-dimethyl-1-methylene-
1,2,3,4-tetrahydronaphthalene (300 mg, 1.19 mmol, 60.5% yield).
Step B. 7-Bromo-3,3-dimethyl-1-methylene-1,2,3,4-tetrahydronaph-
thalene (300 mg, 1.19 mmol) was diluted with diethyl ether (3 mL)
followed by the addition of silver cyanate (537 mg, 3.58 mmol). The
mixture was placed under nitrogen and cooled to 0 °C. I2 (303 mg,
1.19 mmol) was added, and the mixture was stirred for 1 h, warming to
ambient temperature. The mixture was filtered through glass
microfiber paper and concentrated. The residue was diluted with
acetone (3 mL) and NH4OH (600 μL). The mixture was stirred
overnight. The mixture was diluted with ethyl acetate and water. The
layers were separated, and the organics were dried over MgSO4,
filtered, and concentrated. The material was purified on reverse phase
HPLC, eluting with 5−95% acetonitrile/water (0.1% TFA) monitor-
ing at 220 nm to afford 7-bromo-3,3-dimethyl-3,4-dihydro-2H,5′H-
spiro[naphthalene-1,4′-oxazol]-2′-amine (70 mg, 0.226 mmol, 19%
yield).
(R)-2′-Amino-6-(5-chloropyridin-3-yl)-1′,2,2-trimethylspiro-
[chroman-4,4′-imidazol]-5′(1′H)-one ((R)-34). The racemic com-
pound 34 was separated by SFC chromatography using a Chrialpak
AD-H (2 cm × 15 cm) 901061 column. The column was eluted with
15% ethanol (0.2% DEA)/CO2 at 100 mL/min (100 bar). This
afforded complete separation of the single enantiomer (R)-34 (>99%
ee). 1H NMR (400 MHz, CDCl3-d) δ 8.57 (d, J1 = 1.95 Hz, 1H), 8.48,
(d, J1 = 2.34 Hz, 1H), 7.71 (t, J1 = 2.15 Hz, 1H), 7.37 (dd, J1 = 8.59
Hz, J2 = 2.34 Hz, 1H), 6.97 (d, J1 = 8.59 Hz, 1H), 6.94 (s, 1H), 3.21 (s,
3H), 2.55 (d, J1 = 14.1 Hz, 1H), 1.97 (d, J1 = 14.1 Hz, 1H), 1.53 (s,
3H), 1.43 (s, 3H); 13C NMR (500 MHz, CDCl3-d) δ 179.5 (C), 155.7
(C), 154.5 (C), 146.6 (CH), 145.7 (CH), 137.3 (C), 133.6 (CH),
132.0 (C), 129.2 (C), 128.8 (CH), 125.9 (CH), 121.4 (C), 119.6
(CH), 74.3 (C), 64.5 (C), 43.9 (CH2), 29.8−25.0 (CH3), 26.1 (CH3);
LCMS (APCI+) m/z 371.2 (M + 1).
(R)-3-(2-Amino-1,3′,3′-trimethyl-5-oxo-1,3′,4′,5-tetrahydro-
2′H-spiro[imidazole-4,1′-naphthalene]-7′-yl)benzonitrile ((R)-
45). The racemic compound 45 was separated by SFC chromatog-
raphy using a Chiralcel OD-H (3 cm × 15 cm) 07-8754 column,
eluting with 40% methanol (0.1% DEA)/CO2 at 100 bar at a flow rate
of 80 mL/min (injection volume 2 mL, 53 mg/mL methanol). This
1
afforded the single enantiomer (R)-45 (>99% ee). H NMR (400
MHz, CDCl3-d) δ 7.74 (d, J = 1.56 Hz, 1H), 7.69 (d, J = 7.83 Hz, 1H),
7.61−7.58 (m, 1H), 7.50 (t, J = 7.83 Hz, 1H), 7.39 (dd, J1 = 7.83 Hz,
J2 = 2.25 Hz, 1H), 7.21 (d, J = 7.83 Hz, 1H), 7.06 (d, J = 1.56 Hz, 1H),
3.21 (s, 3H), 2.83 (d, J = 15.65 Hz, 1H), 2.61 (dd, J1 = 16.43 Hz, J2 =
2.35 Hz, 1H), 2.29 (d, J = 14.09 Hz, 1H), 1.81 (dd, J1 = 14.09 Hz, J2 =
2.35 Hz, 1H), 1.17 (s, 3H0, 1.02 (s, 3H); 13C NMR (500 MHz,
CDCl3-d) δ 178.5 (C), 156.0 (C), 141.8 (C), 137.8 (C), 137.4 (C),
134.5 (C), 131.5 (CH), 130.7 (CH), 130.7 (CH), 130.7 (CH), 129.6
(CH), 127.3 (CH), 125.3 (CH), 118.8 (C), 112.9 (C), 65.8 (C), 47.0
(CH2), 43.4 (CH2), 31.1−25.9 (CH3), 29.6 (C), 25.9 (CH3). LCMS
(APCI+) m/z 359 (M + 1), retention time 2.263 min.
Step C. 7-Bromo-3,3-dimethyl-3,4-dihydro-2H,5′H-spiro-
[naphthalene-1,4′-oxazol]-2′-amine (25 mg, 0.081 mmol) and 2-
fluoropyridin-3-ylboronic acid (23 mg, 0.16 mmol) were diluted with
dioxane (500 μL) followed by the addition of Pd(PPh3)4 (0.93 mg,
0.000 81 mmol) and Na2CO3 (121 μL, 0.24 mmol). The mixture was
purged with argon, sealed, heated to 85 °C, and stirred for 12 h. The
mixture was loaded onto silica gel and eluted with 1−10% methanol/
DCM (1% NH4OH) to yield 7-(2-fluoropyridin-3-yl)-3,3-dimethyl-
3,4-dihydro-2H,5′H-spiro[naphthalene-1,4′-oxazol]-2′-amine (25 mg,
(R)-2-Amino-7′-(2-fluoropyridin-3-yl)-1,3′,3′-trimethyl-3′,4′-
dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one ((R)-
46). The title compound was prepared according to the procedures
of compound (R)-50, in which 2-fluoropyridin-3-ylboronic acid was
1
used in place of pyrimidin-5-ylboronic acid in step B. H NMR (400
1
MHz, CDCl3-d) δ 8.16 (dd, J1 = 7.04 Hz, J2 = 1.56 Hz, 1H), 7.80−7.75
(m, 1H), 7.42 (dt, J1 = 7.83 Hz, J2 = 1.56 Hz,1H), 7.26−7.23 (m, 1H),
7.27 (d, J = 7.82 Hz, 1H), 7.10 (s, 1H), 3.20 (s, 3H), 2.82 (d, J = 15.65
Hz, 1H), 2.62 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.31 (d, J = 14.09
Hz, 1H), 1.83 (dd, J1 = 14.09 Hz, J2 = 3.13 Hz, 1H), 1.17 (s, 3H), 1.03
(s, 3H). 13C NMR (500 MHz, CDCl3-d) δ 177.1 (C), 160 (C), 157.8
(C), 146.4 (CH), 140.5 (CH), 137.7 (C), 132.7 (C), 132.7 (C), 130.5
(CH), 129.3 (CH), 126.8 (CH), 123 (C), 121.8 (CH), 65.1 (C), 46.8
(CH2), 43.2 (CH2), 31.2−25.8 (CH3), 29.5 (C), 25.8 (CH3). LCMS
(APCI+) m/z 353.2 (M + 1), retention time 2.099 min.
(R)-2-Amino-1,3′,3′-trimethyl-7′-(pyrimidin-5-yl)-3′,4′-dihy-
dro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-one. ((R)-50).
Step A. SFC separation of racemic 2-amino-7′-bromo-1,3′,3′-
trimethyl-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-
one (133 g, 397 mmol) was performed on a Lux Cellulose-4 (3 × 25
cm) column, eluting with 35% methanol (0.1%NH4OH)/CO2 at 100
bar at a flow rate of 200 mL/min (injection volume 2 mL, 309 mg/mL
methanol). The peaks isolated were analyzed on a Lux Cellulose-4
(0.46 cm × 5 cm, 3 μm) column, eluting with 25% methanol (0.1%
NH4OH)/CO2 at 120 bar (flow rate 5 mL/min, 210 nm). From this
separation, (R)-2-amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-
spiro[imidazole-4,1′-naphthalen]-5(1H)-one (peak 1, 46.55 g, chem-
ical purity >99%, >99% ee) was isolated.
0.077 mmol, 95% yield). H NMR (400 MHz, CDCl3-d) δ 8.17 (m,
1H), 7.85 (m, 1H), 7.44 (s, 1H), 7.35 (m, 1H), 7.23 (m, 1H), 7.11 (d,
J = 7.8 Hz, 1H), 4.37 (s, 2H), 2.7 (d, J = 16 Hz, 1H), 2.56 (d, J = 16
Hz, 1H), 2.05 (d, J = 13.7 Hz, 1H), 1.84 (d, J = 13.7 Hz, 1H), 1.1 (s,
3H), 0.97 (s, 3H); LCMS (APCI+) m/z 326.1 (M + 1).
3,3-Dimethyl-7-(pyrimidin-5-yl)-3,4-dihydro-2H,5′H-spiro-
[naphthalene-1,4′-oxazol]-2′-amine (rac-63). rac-63 was synthe-
sized in the same fashion as compound 62, replacing 2-fluoropyridin-3-
ylboronic acid with pyrimidin-5-ylboronic acid to afford 3,3-dimethyl-
7-(pyrimidin-5-yl)-3,4-dihydro-2H,5′H-spiro[naphthalene-1,4′-oxa-
1
zol]-2′-amine (18 mg, 0.058 mmol, 72% yield). H NMR (400 MHz,
CDCl3-d) δ 9.2 (s, 1H), 8.9 (s, 2H), 7.46 (d, J = 1.96 Hz, 1H), 7.37
(dd, J1 = 7.8 Hz, J2 = 1.96 Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 4.35 (dd,
J1 = 8.2 Hz, J2 = 4.3 Hz, 2H), 2.7 (d, 16.4 Hz, 1H), 2.57 (dd, J1 = 16.4
Hz, J2 = 1.6 Hz, 1H), 2.06 (d, J = 13.7 Hz, 1H), 1.86 (dd, J1 = 13.7 Hz,
J2 = 1.6 Hz, 1H), 1.1 (s, 3H), 0.98 (s, 3H); 13C NMR (500 MHz,
CDCl3-d) δ 160.2 (C), 157.3 (CH), 154.8 (CH), 154.8 (CH), 142.5
(C), 136.8 (C), 134.3 (C), 132.8 (C), 130.2 (CH), 125.8 (CH), 125.7
(CH), 83.0 (CH2), 69.6 (C), 50.6 (CH2), 44.1 (CH2), 30.5 (C),
30.3−27.5 (CH3); LCMS (APCI+) m/z 309.1 (M + 1).
7-(5-Fluoropyridin-3-yl)-3,3-dimethyl-3,4-dihydro-2H,5′H-
spiro[naphthalene-1,4′-oxazol]-2′-amine (rac-61). rac-61 was
synthesized in the same fashion as compound 62, substituting 2-
fluoropyridin-3-ylboronic acid for (5-fluoropyridin-3-yl)boronic acid to
afford 7-(5-fluoropyridin-3-yl)-3,3-dimethyl-3,4-dihydro-2H,5′H-spiro-
[naphthalene-1,4′-oxazol]-2′-amine (10 mg, 0.031 mmol, 38% yield).
1H NMR (400 MHz, CDCl3-d) δ 8.65 (m, 1H), 8.42 (d, J = 2.7 Hz,
Step B. (R)-2-Amino-7′-bromo-1,3′,3′-trimethyl-3′,4′-dihydro-2′H-
spiro[imidazole-4,1′-naphthalen]-5(1H)-one (1.00 g, 2.97 mmol),
pyrimidin-5-ylboronic acid (0.479 g, 3.87 mmol), and Pd(PPh3)4
(0.0859 g, 0.0744 mmol) were combined with 15 mL of dioxane
and 2 M Na2CO3 (3.72 mL, 7.44 mmol) (both degassed with nitrogen
sparge for 30 min prior to use), and the reaction mixture was heated in
1H), 7.55 (m, 1H), 7.45 (d, J1 = 1.56 Hz, 1H), 7.35 (dd, J1 = 7.8 Hz, J2
= 1.9 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 4.35 (dd, J1 = 8.2 Hz, J2 = 4.3
U
dx.doi.org/10.1021/jm4002154 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a 100 °C reaction block and stirred for 17 h. The reaction mixture was
then concentrated, and the residue was combined with ethyl acetate
and water. The mixture was extracted 2× with ethyl acetate, and the
combined extracts were dried (Na2SO4), filtered, and concentrated.
The crude was purified on silica gel (5−20% MeOH in dichloro-
methane gradient) to give (R)-2-amino-1,3′,3′-trimethyl-7′-(pyrimi-
din-5-yl)-3′,4′-dihydro-2′H-spiro[imidazole-4,1′-naphthalen]-5(1H)-
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Andrew Allen (Array BioPharma) for multidimen-
sional NMR spectroscopy support and chiral HPLC methods
development. We thank Chris Hamman (Genentech) and
Mengling Wong (Genentech) for chiral separation.
1
one (0.755 g, 2.25 mmol, 75.7% yield) as a white powder. H NMR
(400 MHz, CDCl3-d) δ 9.16 (s, 1H), 8.85 (s, 2H), 7.42 (dd, J = −7.83
Hz, J2 = 2.35 Hz, 1H), 7.09 (d, J = 1.56 Hz, 1H), 3.22 (s, 3H), 2.86 (d,
J = 15.65 Hz, 1H), 2.63 (dd, J1 = 16.43 Hz, J2 = 2.35 Hz, 1H), 2.29 (d,
14.08 Hz, 1H), 1.84 (dd, J1 = 14.09 Hz, J2 = 2.35 Hz, 1H), 1.18 (s,
3H), 1.03 (s, 3H). 13C NMR (500 MHz, CDCl3-d) δ 178.0 (C), 157.5
(CH), 155.7 (C), 154.8 (CH), 154.8 (CH), 138.1 (C), 135.3 (C),
133.8 (C), 133.1 (C), 131.1 (CH), 126.9 (CH), 125.3 (CH), 66.0
(C), 46.8 (CH2), 43.4 (CH2), 31.1−26.0 (CH3), 29.6 (C), 26.0
(CH3); LCMS (APCI+) m/z 336.2 (M + 1)+, retention time 1.905
min (5 min run).
(R)-3,3-Dimethyl-7-(pyrimidin-5-yl)-3,4-dihydro-2H,5′H-
spiro[naphthalene-1,4′-oxazol]-2′-amine ((R)-63). The racemic
compound 63 was separated by SFC chromatography using a chiral
column (Chiralpak AD-H, 2 cm × 15 cm), eluting with 35% methanol
(20 nM NH3)/CO2, 100 bar, 60 mL/min and monitored at 220 nm to
afford the title compound (>99% ee). 1H NMR (400 MHz, CDCl3-d)
δ 9.2 (s, 1H), 8.9 (s, 2H), 7.46 (d, J = 1.96 Hz, 1H), 7.37 (dd, J1 = 7.8
Hz, J2 = 1.96 Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 4.35 (dd, J1 = 8.2 Hz,
J2 = 4.3 Hz, 2H), 2.7 (d, 16.4 Hz, 1H), 2.57 (dd, J1 = 16.4 Hz, J2 = 1.6
Hz, 1H), 2.06 (d, J = 13.7 Hz, 1H), 1.86 (dd, J1 = 13.7 Hz, J2 = 1.6 Hz,
1H), 1.1 (s, 3H), 0.98 (s, 3H); 13C NMR (500 MHz, CDCl3-d) δ
160.2 (C), 157.3 (CH), 154.8 (CH), 154.8 (CH), 142.5 (C), 136.8
(C), 134.4 (C), 132.7 (C), 130.2 (CH), 125.8 (CH), 125.6 (CH),
83.0 (CH2), 69.5 (C), 50.6 (CH2), 44.0 (CH2), 30.5 (C), 30.3−27.4
(CH3); LCMS (APCI+) m/z 309.1 (M + 1).
ABBREVIATIONS USED
■
CatD, cathepsin D; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride; LLC-PK1, Lilly Laboratories cell
porcine kidney; HTRF, homogeneous time-resolved fluores-
cence; fu, fraction unbound; CL, clearance; CLu, unbound
clearance; Cfree, unbound concentration; Pi (e.g., P3, P2′),
amino acid residue from N to C terminus of the polypeptide
substrate; Si (e.g., S3, S2′), binding subsite
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ASSOCIATED CONTENT
Accession Codes
■
The PDB accession codes for the X-ray cocrystal structures of
BACE1 + 1a, BACE1 + 12, BACE1 + 13, and BACE1 + (R)-50
are 4JOO, 4JP9, 4JPC, and 4JPE, respectively.
AUTHOR INFORMATION
Corresponding Author
*Phone: 303-386-1153. Fax: 303-386-1130. E-mail: kevin.
hunt@arraybiopharma.com.
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