Inhibitors of β-site amyloid precursor protein cleaving enzyme (BACE1): Identification of (S)-7-(2-fluoropyridin-3-yl)-3-((3-methyloxetan-3-yl)ethynyl)-5′ H -spiro[chromeno[2,3- b ]pyridine-5,4′-oxazol]-2′-amine (AMG-8718)

Article abstract of DOI:10.1021/jm5012676

We have previously shown that the aminooxazoline xanthene scaffold can generate potent and orally efficacious BACE1 inhibitors although certain of these compounds exhibited potential hERG liabilities. In this article, we describe 4-aza substitution on the xanthene core as a means to increase BACE1 potency while reducing hERG binding affinity. Further optimization of the P3 and P2′ side chains resulted in the identification of 42 (AMG-8718), a compound with a balanced profile of BACE1 potency, hERG binding affinity, and Pgp recognition. This compound produced robust and sustained reductions of CSF and brain Aβ levels in a rat pharmacodynamic model and exhibited significantly reduced potential for QTc elongation in a cardiovascular safety model.

Full text of DOI:10.1021/jm5012676

Article
pubs.acs.org/jmc
Inhibitors of β‑Site Amyloid Precursor Protein Cleaving Enzyme
(BACE1): Identification of (S)‑7-(2-Fluoropyridin-3-yl)-3-((3-
methyloxetan-3-yl)ethynyl)-5′H‑spiro[chromeno[2,3‑b]pyridine-5,4′-
oxazol]-2′-amine (AMG-8718)
Thomas A. Dineen,*,† Kui Chen,§ Alan C. Cheng,§ Katayoun Derakhchan,⊥ Oleg Epstein,† Joel Esmay,∥
Dean Hickman,∥ Chuck E. Kreiman,† Isaac E. Marx,† Robert C. Wahl,§ Paul H. Wen,‡
Matthew M. Weiss,† Douglas A. Whittington,§ Stephen Wood,‡ Robert T. Fremeau, Jr.,‡ Ryan D. White,†,‡
and Vinod F. Patel†,#
‡
§
∥
†Departments of Therapeutic Discovery, Neuroscience, Molecular Structure and Characterization, Pharmacokinetics and Drug
Metabolism, and ⊥Comparative Biology and Safety Sciences, Amgen, Inc., 360 Binney Street, Cambridge, Massachusetts 02142, and
One Amgen Center Drive, Thousand Oaks, California 91320, United States
S
* Supporting Information
ABSTRACT: We have previously shown that the amino-
oxazoline xanthene scaffold can generate potent and orally
efficacious BACE1 inhibitors although certain of these
compounds exhibited potential hERG liabilities. In this article,
we describe 4-aza substitution on the xanthene core as a means
to increase BACE1 potency while reducing hERG binding
affinity. Further optimization of the P3 and P2′ side chains
resulted in the identification of 42 (AMG-8718), a compound
with a balanced profile of BACE1 potency, hERG binding
affinity, and Pgp recognition. This compound produced robust and sustained reductions of CSF and brain Aβ levels in a rat
pharmacodynamic model and exhibited significantly reduced potential for QTc elongation in a cardiovascular safety model.
BACE1 inhibitors produced large, polar compounds with
unfavorable physicochemical properties and consequently poor
pharmacokinetics and central nervous system (CNS) pene-
tration.6 More recently, a number of groups have described
BACE1 inhibitors that employ a 2-aminoheterocycle which
engages in hydrogen bonding interactions with the catalytic
aspartic acid residues of the protease.7 These heterocyclic units
bind to the enzyme in an orientation that allows for further
extension of a side chain into the S1 and S3 pockets. These
designs have resulted in the identification of a number of potent
BACE1 inhibitors with significantly improved pharmacokinetic
profiles and CNS penetration compared with earlier
peptidomimetic inhibitors.
Our own efforts in this area led to the discovery of the
aminooxazoline xanthene scaffold,8 which enabled extension of
side chains into both the S3 and S2′ pockets of BACE1 and led
to the identification of compound 1 (Figure 1).9 While this
compound led to robust reduction in central Aβ when dosed
orally to Sprague−Dawley (SD) rats, its advancement was
limited due to off-target activity on the hERG channel. Indeed,
when evaluated in an anesthetized dog cardiovascular model,
compound 1 caused prolongation of the QTc interval at
INTRODUCTION
■
Alzheimer’s disease (AD) is the most common form of
dementia and is characterized by cognitive decline, changes in
thinking, memory loss, and ultimately death. Although AD
affects over 18 million people worldwide, there are currently no
available disease-modifying therapies and the available treat-
ments provide only limited benefit.1
A large body of evidence has implicated the formation and
accumulation of amyloid β (Aβ) peptides, specifically Aβ40 and
Aβ42, in the brains of AD patients as critical factors in the
pathogenesis of the disease.2 Aβ peptides are formed in the
brain via sequential proteolytic cleavage of amyloid precursor
protein (APP) by the aspartyl proteases BACE1 (β-site APP
cleaving enzyme) and γ-secretase.3 The amyloid hypothesis
suggests that inhibiting the formation and accumulation of Aβ
in the brain could result in a disease-modifying effect in AD
patients.4 Recently published genetic evidence has provided
further support for this approach, suggesting that even modest
decreases in the formation of Aβ (as a result of a mutation in
the gene encoding APP) may confer significant protection
against the development of AD.5
Since its identification in 1999,3 BACE1 has attracted a
significant amount of attention as a potential target for the
development of an AD therapy. BACE1 contains a long,
shallow, and hydrophilic active site, and early efforts to identify
Received: August 18, 2014
© XXXX American Chemical Society
A
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Figure 1. Previous efforts to balance hERG inhibition and Pgp-mediated efflux.
Scheme 1. Synthesis of the P2′-Neopentyloxy 4-Azaxanthenes 26−28
a
a
Reagents: (a) (i) NaH, DMF, 130 °C, 55%, (ii) PPA, 140 °C, 93%; (b) (i) MeMgCl, THF, −30 °C, HCl/Et2O, (ii) I2, AgOCN, THF, −40 °C, (iii)
NH3, 23 °C; (iv) chiral SFC, 29%; (c) BBr3, DCM, 0 °C, 51%; (d) neopentyl iodide, Cs2CO3, DMF, 100 °C, 63%; (e) Ar-B(OH)2, Pd(PPh3)4 or
Pd(AmPhos)2Cl2, K2CO3, THF−H2O, 110 °C, 72−90%.
a
Scheme 2. Preparation of 4-Azaxanthenes via Late-Stage Installation of the P2′ Group
a
Reagents: (a) (i) NaH, DMF, 130 °C, 76%, (ii) PPA, 140 °C, 85%; (b) (i) MeMgCl, THF, −30 to 0 °C, THF−CHCl3, 80 °C, (ii) I2, AgOCN,
THF, −20 °C, (iii) NH3, 23 °C, (iv) chiral SFC, 13%; (c) 2-fluoro-3-pyridineboronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane−water, 70 °C, 91%; (d)
R-B(OH)2, Pd(PPh3)4, K2CO3, 1,4-dioxane−water, 80 °C, 40−59%; (e) R-CCH, Pd(PPh3)4, CuI, i-PrNH2−DMF, 90 °C, 58−95%; (f) R-CC-
TMS, Pd(PPh3)4, CuI, TBAF, THF, 70 °C, 89% for 42.
relatively low exposure multiples over the in vivo EC50 for brain
Aβ reduction in a rat pharmacodynamic model. Subsequent
SAR studies demonstrated that the hERG binding affinity of
these compounds could be reduced by incorporating polar
heteroatoms in the P2′ region of the inhibitor, albeit at the
expense of increased Pgp-mediated efflux and consequently
lower CNS exposure.10 Further fine-tuning of physicochemical
properties indicated that these increases in transporter-
mediated efflux could be attenuated by reducing the PSA of
the P3 group and by fluoro-substitution on the xanthene ring.
The incorporation of a fluorine atom at the 4-position of the
xanthene ring (e.g., 2) also resulted in compounds with
improved (5−10×) BACE1 potency.9 The increased BACE1
potency within this series was attributed to a hydrogen bonding
interaction between the 4-fluoro atom and Trp76 of BACE1, as
observed in crystal structures of these compounds with the
enzyme.11
In this article, an alternative strategy is described for
balancing BACE1 potency, hERG binding affinity, and Pgp-
mediated efflux by incorporating a nitrogen atom into the
xanthene core structure of the aminooxazoline xanthene
inhibitors. Further optimization of the P3 and P2′ groups led
to the identification of a metabolically stable, orally efficacious
BACE1 inhibitor with a favorable balance of in vivo efficacy to
off-target effects.
CHEMISTRY
■
The synthesis of inhibitors BACE1 inhibitors 26−28 (Table 1)
is outlined in Scheme 1. Coupling of p-bromophenol (3) with
2-fluoropyridine 4 under basic conditions in DMF, followed by
ring-closure of the resulting intermediate in neat polyphos-
B
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a
Scheme 3. Synthesis of 4-Azaxanthenes 33−37
a
Reagents: (a) (i) NaH, DMF, 140 °C, 83%, (ii) PPA, 140 °C, 66%; (b) (i) MeMgBr, THF, −30 to 0 °C, CH3OH, 60 °C, 53%, (ii) I2, AgOCN,
THF, −20 °C, (iii) NH3, 23 °C, (iv) chiral SFC, 28%; (c) BBr3, DCM, 0 °C, 62%; (d) (i) 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane, Pd(PPh3)4, K2CO3, DMF, water, 80 °C, 70%, (ii) H2 (1 atm), 10% Pd/C, MeOH, 82% of 18a; (e) R-B(OH)2, Pd(PPh3)4 or
Pd(AmPhos)2Cl2, K2CO3, 1,4-dioxane−water, 75−100 °C, 34−69% for 18b, 18d, and 18e; (f) morpholine, Pd2(dba)3, DavePhos, LHMDS, THF,
70 °C, 51% of 18c; (g) PhNTf2, Et3N, DCM, 67−89%; (h) 2-fluoro-3-pyridineboronic acid, Pd(PPh3)4, K2CO3, 1,4-dioxane−water, 80 °C, 34−97%.
a
Scheme 4. Synthesis of BACE1 Inhibitor 30
a
Reagents: (a) (i) LiTMP, THF, −10 °C, then CO2(g), 20, K2CO3, DMF, 140 °C, (ii) PPA, 140 °C; (b) (i) Pd(OAc)2, X-Phos, B2(pin)2, K3PO4,
1,4-dioxane, 23 °C, (ii) 30% H2O2, THF, 66%; (c) (i) 3-bromo-2-methylpropene, DBU, DCM, (ii) NIS, MeOH, (iii) MeMgCl, THF, −40 °C, (iv)
LiBEt3H, THF, 9% over four steps; (d) (i) HCl/1,4-dioxane, 50 °C, (ii) I2, AgOCN, THF, −20 °C, (iii) NH3, 23 °C, 16%; (e) (i) 2-fluoro-3-
pyridineboronic acid, Pd(PPh3)4, K2CO3, THF−water, 110 °C, (ii) chiral SFC, 16%.
phoric acid, afforded xanthenone 5. As described in the
preceding article for the analogous 4-fluoroxanthenes,9 the
aminooxazoline unit was installed via a three-step sequence as
follows. Reaction of ketone 5 with MeMgCl and subsequent
HCl treatment afforded an unstable exo-olefin. The olefin was
treated directly with in situ generated iodine isocyanate
followed by a solution of ammonia in 2-propanol to yield the
2-aminooxazoline xanthene in racemic form.12 Separation of
the racemate through chiral supercritical fluid chromatography
(SFC) afforded the (S)-enantiomer 6 on multigram scale. The
preparation of neopentyl ether 8 was accomplished by boron
tribromide-mediated demethylation of 6, followed by cesium
carbonate-promoted alkylation of intermediate phenol 7 with
neopentyl iodide. Palladium catalyzed Suzuki cross-coupling
reactions with the corresponding boronic acids13 afforded the
final analogues 26−28.
The compounds in Tables 2 and 3 were generated via several
different routes. Selected C-linked P2′ analogues (31, 32, and
38−42) were prepared as described in Scheme 2 via the
common dihalo intermediate 12. Coupling of p-iodophenol (9)
and 2-chloropyridine 10, followed by acidic ring closure, gave
xanthenone 11. Formation of the 2-aminooxazoline xanthene
unit and chiral separation was accomplished as described above
to afford the bis-functionalized iodo-bromo intermediate 12.
C
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a
Scheme 5. Synthesis of BACE1 Inhibitor 29
a
Reagents: (a) BBr3, DCM, 0 °C, 99%; (b) neopentyl iodide, Cs2CO3, DMF, 100 °C, 48%; (c) (i) 2-fluoro-3-pyridineboronic acid, Pd(PPh3)4,
K2CO3, THF−water, sealed tube, 90 °C, (ii) chiral SFC, 24%.
Table 1. Evaluating Aza-substitution in the Xanthene Core
a
b
c
Values represent the mean values of at least two experiments SD. Human embryonic kidney cells. Apparent permeability measured in parental
LLC-PK1 cells. Values are an average of apical to basolateral (A to B) and basolateral to apical (B to A) velocities and are reported as 10−6 cm/s.
d
e
Efflux measured in LLC-PK1 cells transfected with either rat MDR1A/1B or human MDR1 and are reported as a ratio of (B to A)/(A to B). Rat
liver microsomal (RLM) and human liver microsomal (HLM) clearance. Compound concentration = 1 μM. Microsomal protein concentration =
250 μg/mL.
Selective Suzuki cross-coupling of the aryl iodide with 2-fluoro-
3-pyridineboronic acid provided the penultimate bromide 13,
which was subjected to either a second Suzuki cross-coupling or
a Sonogashira cross-coupling to generate the desired
compounds in good overall yield.
Alternatively, other analogues (33−37) in Table 2 were
prepared by initial coupling of the P2′ group followed by a final
introduction of the 2-fluoro-3-pyridyl P3 group as described in
Scheme 3. For these examples, phenol 14 and 2-chloropyridine
10 were converted to aminooxazoline 16 under the previously
described conditions. Boron tribromide-mediated demethyla-
tion afforded bromophenol 17, which served as a versatile
intermediate for diverse Pd-catalyzed C,C and C,N cross-
coupling reactions to install the P2′ group. Triflation of the
phenols 18a−e afforded triflates 19a−e, which were subjected
to a final Suzuki cross-coupling with 2-fluoro-3-pyridineboronic
acid, yielding the desired compounds 33−37.
The BACE1 inhibitor 30 was prepared via a separate route
shown in Scheme 4. Xanthenone 21 was synthesized starting
from bromophenol 3 and fluoropyridine 20 and was converted
to bromophenol 22 via a two-step borylation-oxidation
protocol. Formation of the 2-methoxy-2-methylpropoxy side
chain of 24 was accomplished in four steps beginning with
alkylation of the phenol with 3-bromo-2-methylpropene. The
resulting alkene underwent iodo-etherification in the presence
of NIS in methanol. Sequential treatment of the product with
D
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Table 2. P2′ SAR of 4-Azaxanthenes
a
b
c
Values represent the mean values of at least two experiments SD. Human embryonic kidney cells. Apparent permeability measured in parental
LLC-PK1 cells. Values are an average of apical to basolateral (A to B) and basolateral to apical (B to A) velocities and are reported as 10−6 cm/s.
d
e
Efflux measured in LLC-PK1 cells transfected with either rat MDR1A/1B or human MDR1 and are reported as a ratio of (B to A)/(A to B). Rat
liver microsomal (RLM) and human liver microsomal (HLM) clearance. Compound concentration = 1 μM. Microsomal protein concentration =
250 μg/mL.
MeMgCl and lithium triethylborohydride in a single pot
afforded the alcohol 23. Exposure of 23 to acid promoted
elimination of the alcohol to form the exo-olefin, which was
converted to the aminooxazoline 24. A final Suzuki cross-
coupling with 2-fluoro-3-pyridineboronic acid was followed by
chiral separation to furnish compound 30.
E
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Table 3. Optimization of the P2′ Alkyne
a
b
c
Values represent the mean values of at least two experiments SD. Human embryonic kidney cells. Apparent permeability measured in parental
LLC-PK1 cells. Values are an average of apical to basolateral (A to B) and basolateral to apical (B to A) velocities and are reported as 10−6 cm/s.
d
e
Efflux measured in LLC-PK1 cells transfected with either rat MDR1A/1B or human MDR1 and are reported as a ratio of (B to A)/(A to B). Rat
liver microsomal (RLM) and human liver microsomal (HLM) clearance. Compound concentration = 1 μM. Microsomal protein concentration =
250 μg/mL.
The regioisomeric azaxanthene compound 29 (Table 1) was
prepared as shown in Scheme 5. The racemic intermediate 16
(Scheme 1) was subjected to treatment with boron tribromide
to give the corresponding phenol, which underwent a
subsequent alkylation to form neopentyl ether 25. Suzuki
cross-coupling with 5-pyrimidyl boronic acid, followed by chiral
separation, yielded the desired isomer 29.
indicating that compound 26 is a Pgp-substrate. Reducing the
overall PSA of the molecule by changing the P3 group from a 5-
pyrimidyl to a 3-pyridyl ring (27), isomeric with compound 1,
returned the favorable efflux properties of 1 while maintaining
low hERG activity. Replacement of the P3 group with the 2-
fluoro-3-pyridyl ring (28) generated a compound with excellent
BACE1 potency in our enzymatic and functional assays, low
efflux ratios, and moderate hERG binding. We also examined
incorporation of a nitrogen atom at the 5-position of the
xanthene. Compound 29 showed dramatically reduced BACE1
potency in the enzymatic and functional assays, suggesting an
unfavorable interaction of this atom with the protein.
Interestingly, incorporation of a nitrogen atom at the 4-
position led to decreases in microsomal stability when coupled
with either a 5-pyrimidine (26) or a 3-pyridly group (27) in P3,
however, the 2-fluoro-3-pyridyl analogue (28) maintained the
low metabolism observed with all-carbon analogue 1.
As mentioned before, compound 28 demonstrated improved
BACE1 functional potency and decreased hERG activity,
relative to 1, while maintaining the high permeability, low
Pgp efflux, and good microsomal stability−properties consistent
with good distribution into the CNS. In an effort to further
reduce hERG activity within this series, additional P2′ side
chains were explored. As shown in Table 2, a number of groups
which had previously been identified as beneficial for BACE1
potency9 were incorporated into the P2′ side chain of the 4-
azaxanthene aminooxazolines. Addition of a methoxy group to
the neopentyloxy P2′ side chain (30) resulted in a slight
decrease of BACE1 enzymatic and functional potencies.
Inhibitors containing a dihydropyran P2′ group (31, 32, and
34) were all approximately 2-fold less potent than 28, and the
fully saturated tetrahydropyran 33 was approximately 8-fold less
potent in the functional assay. Similarly, the N-morpholino
RESULTS AND DISCUSSION
■
Our previous work on aminooxazoline xanthene BACE1
inhibitors had demonstrated that incorporation of a fluorine
atom in the xanthene core, especially at C(4), afforded a
significant increase in enzymatic potency.9 The enhanced
potency was believed to arise from a favorable hydrogen-
bonding interaction between the fluorine atom and NHTrp76
of the protein, and we hypothesized that a nitrogen atom at the
4-position of the xanthene could similarly engage Trp76.
Furthermore, employing a nitrogen atom to exploit this
interaction with the protein could not only potentially boost
potency against BACE1 but at the same time reduce hERG
binding affinity as a result of the increase in PSA.14 While there
was a possibility that the additional PSA could result in
increased transporter-mediated efflux,15 the consequential
decreased exposure in the CNS could potentially be offset by
increases in BACE1 potency, enabling the discovery of BACE1
inhibitors with good in vivo efficacy and improved
cardiovascular safety margins.
We began our investigation by preparing 26, the 4-aza
analogue of one of our initial leads, 1. As shown in Table 1, the
4-azaxanthene core did indeed afford both greater enzymatic
and functional potency than the all-carbon analogue 1. As
expected, the higher PSA led to a hERG binding Ki >10 μM in
addition to a slight increase in Pgp-mediated efflux ratios to >2,
F
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Figure 2. Co-crystal structure of 42 with BACE1. (a) Interactions of 42 with the catalytic aspartic acids, Ser229, and Trp76 of BACE1. (b)
Interaction of the P2′ oxetane unit with Arg128 in S2′.
Table 4. Reduction of CSF Aβ40 in Wild-Type Sprague−Dawley Rats
a
b
b
c
c
d
compd
CSF Aβ40 reduction (%)
brain Aβ40 reduction (%)
[plasma]total (μM)
[plasma]unbound (μM)
[CSF] (μM)
[CSF]/cell IC50
34
42
53
69
16
48
1.01
4.43
0.045
0.059
0.005
0.017
0.75
3.9
a
b
Compounds were dosed orally at 10 mg/kg as a solution (2% HMPC, 1% Tween80) to male rats. n = 5 animals per group. CSF and brain Aβ40
c
d
measured at t = 4 h. Plasma drug concentration measured at t = 4 h. CSF drug concentration measured at t = 4 h.
substituent in compound 35 resulted in reduced BACE1
activity in both the enzymatic and functional assays. While
compounds with aryl groups in P2′ (36−38) maintained good
potency in the BACE1 enzymatic assay, they were consistently
less potent (approximately 3−6-fold) in the functional assay.
Finally, alkynes 39 and 40 demonstrated no enzyme-to-cell
shift and consequently produced low nanomolar inhibitors in
the functional cellular assay.
Most compounds with a polar heteroatom incorporated in
the P2′ side chain showed significantly decreased hERG
binding affinity (Ki > 10 μM), albeit in some cases at the
expense of increased human and rat Pgp recognition. For
example, Pgp efflux ratios for ethereal compounds 30, 31, 33,
and 35 were >10. Shielding of the ethereal oxygen with methyl
substitution, as in dihydropyran 34, resulted in a favorable
combination of low hERG binding affinity and low Pgp efflux
ratios. The dihydropyran isomer 32 showed modest efflux
when compared with close structural analogues 31 and 33.
Tolyl-substituted inhibitor 36 and aryl nitrile 37 both
maintained significant hERG binding activity, while fluoropyr-
idine 38 had hERG Ki > 10 μM. Compound 37 showed
reduced Pgp efflux activity compared to compound 38.
Incorporation of a methoxy group into the t-butylacetylene
unit of compound 39 yielded a compound with excellent
BACE1 potency, low activity in hERG binding assay, and low
efflux ratios (40).
Most compounds we examined were stable in human and rat
liver microsomes, with the exception of compound 40. Given
the attractive overall profile of 40, we sought to further
understand its high intrinsic clearance with the aim of
generating a more metabolically stable analogue. Metabolite
identification studies on a related analogue with the same P2′
side chain (data not shown), suggested that the methoxy ether
of 40 may undergo oxidative demethylation to form alkynol 41
(Table 3). In support of this hypothesis, alkynol 41 was
independently synthesized and found to be stable in human and
rat liver microsomes. Although this compound also showed
good BACE1 potency and low activity in the hERG binding
assay, it was subject to extremely high Pgp efflux (efflux ratio =
>50 and 44 for human and rat, respectively), likely as a result of
the additional hydrogen bond donor of the free alcohol. To
combine the low intrinsic clearance of alkynol 41 with the low
efflux ratios of methyl ether 40, an inhibitor featuring a 3-
methyl-3-oxetane ethynyl group in P2′ (42) was targeted. The
oxetane group was predicted to be more stable toward oxidative
dealkylation, and it was expected to be less likely a Pgp
recognition element.15 In fact, compound 42 (AMG-8718)
exhibited good stability in human and rat liver microsomes and
only modest Pgp efflux ratios. Importantly, this compound
demonstrated high BACE1 potency, low activity in the hERG
binding assay, and excellent permeability.
An X-ray cocrystal structure of compound 42 with BACE1 is
shown in Figure 2.16 As observed in previous cocrystallized
BACE1 inhibitors, the aminooxazoline unit engages in
hydrogen-bonding interactions with the catalytic aspartic acid
residues of the enzyme, and the nitrogen atom of the 2-pyridyl-
3-fluoro group interacts with Ser229 via a bridging water
molecule in the S3 pocket. As anticipated, the nitrogen atom in
the xanthene ring system engages Trp76 in a hydrogen-bonding
interaction, likely resulting in the enhanced potency observed
for 4-azaxanthenes relative to their all-carbon analogues. An
additional hydrogen-bonding interaction in S2′ was observed
between the oxetane oxygen and Arg128.
A potential concern with compound 42 was the possibility of
CYP inactivation as a result of oxidation of the alkynyl group to
form reactive metabolites,17 however, no time-dependent
inhibition of CYP activity was detected when 42 was examined
in IC50 shift experiments (30 min incubation).18 Additionally,
given the potentially reactive nature of the 2-fluoropyridine and
oxetane functional groups, 42 was incubated in buffered
solutions in the presence of glutathione (GSH), as well as
with hepatocytes, and no incorporation of GSH was observed
under these conditions.
G
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BACE inhibitors 34 and 42 possessed a good overall balance
of properties (BACE1 functional potency, permeability,
intrinsic stability, and Pgp efflux ratios) and were therefore
evaluated in vivo for their ability reduce the concentration of
CNS Aβ in a rat pharmacodynamic model. Male Sprague−
Dawley rats were dosed with compounds 34 or 42 (10 mg/kg)
by oral gavage, where after 4 h plasma and CSF drug levels
were determined along with Aβ40 concentrations in the CSF
and brain.19 As shown in Table 4, both compounds 34 and 42
significantly decreased Aβ40 levels in the CSF at the 4 h time
point (53% and 69%, respectively), however, only 42 produced
a robust response in the brain (48% reduction of Aβ40 levels for
42 vs 16% for 34). The reason for this disparity is potentially
due to a difference in exposure multiples for the two
compounds in the brain. Although both compounds achieved
similar unbound concentrations in plasma at the 4 h time point,
42 achieved an approximately 5-fold higher exposure than 34 in
the CSF compartment, which may reflect a higher unbound
concentration in the brain. However, it is possible that CSF
concentrations may be inflated relative to unbound brain
concentrations as a result of active blood-to-CSF transport by
Pgp.20
a time-course pharmacodynamic study in rat at 30 mg/kg. As
shown in Figure 3, 42 demonstrated robust and sustained
reduction of Aβ levels in both the CSF and brain for over 8 h.
The lowest concentrations of Aβ in both compartments were
observed between 4 and 5 h, and the concentrations of Aβ
returned to predosing levels within 16−20 h. The changes in
brain Aβ concentration tended to precede those in the CSF by
a small margin. Using an indirect response PK/PD model,21
which accounts for the rate of Aβ formation and clearance in
the rat CNS, the in vivo plasma IC50,unbound of 42 was
determined to be 62 nM. The difference between the in vivo
IC50,unbound and the IC50 of 42 in the BACE1 functional assay
(4.4 nM) is likely due, in part, to reduced exposure in the brain
as a result of Pgp efflux.
The effects of 42 in an anesthetized dog cardiovascular safety
model were studied. The compound was administered
intravenously as a series of three 30 min infusions at doses of
2.5, 8, and 16 mg/kg to two chloralose-anesthetized dogs. The
unbound plasma concentrations were assessed at the end of
each infusion and determined (average) to be 0.298, 1.70, and
3.62 μM, respectively. No significant effects on the QTc
interval were observed at these doses. The unbound plasma
concentration at the high dose was 201-fold over the plasma
EC50,unbound for CSF Aβ reduction in rats and 58-fold over the
in vivo IC50,unbound as determined by the indirect response
model. The margins observed in this study represent a
significant improvement over compounds like 1 and 2.
Our strategy of installing a nitrogen atom at the 4-position of
the xanthene to engage Trp76 resulted in the discovery of
BACE1 inhibitors with increased BACE1 potency and reduced
hERG binding affinity. An optimized compound from this
series, 42, showed a 5-fold improvement in BACE1 functional
potency compared with 1, an earlier lead (Figure 4, Table 6).
Although these compounds have similar total PSA, 42 is more
polar (log P = 2.53 for 42 vs log P = 5.20 for 1), which may
account for the significantly lower activity of this compound in
the hERG binding assay.14 The impact of increased Pgp efflux
for 42 observed in the in vitro assays was mitigated by the high
functional potency and permeability of this compound,
resulting in robust reductions in CNS Aβ in the rat
pharmacodynamic model. As a result of the balance of
BACE1 potency, hERG binding affinity, and Pgp recognition
for compound 42, significantly higher safety margins for
elongation of the QTc interval in the dog cardiovascular model
relative the EC50 and IC50 measured in the rat pharmacody-
namic model were achieved.
Finally, compounds 1 and 42 were tested against a panel of
other aspartyl proteases. Both compounds demonstrated
limited selectivity for BACE1 over homologue BACE2, and
the high selectivity for BACE1 over both renin and pepsin
observed with 1 was preserved with 42. Although 1 showed
only modest selectivity for BACE1 against cathepsins D and E
(478× and 139×, respectively), 42 was very selective (3285×
and 1429×, respectively), a result of both increased BACE1
potency and lower cathepsin activity for this compound.
On the basis of the more significant pharmacodynamic
response observed with 42, the pharmacokinetic behavior of
this compound in rat, dog, and cynomolgus monkey was
evaluated. Consistent with its stability in liver microsomes, 42
showed moderate total clearance, moderate Vdss, and half-lives
of ca. 5−8 h across all three species (Table 5). The clearance in
Table 5. Pharmacokinetic Profiles and Plasma Protein
Binding of 42
a
b
iv
po
plasma
protein
Cl
Vdss
t1/2
Cmax
tmax
a
species
rat
(L/h/kg) (L/kg) (h) (μM) (h)
% F binding (Fu)
0.33
0.26
1.1
1.6
4.8
5.2
3.8
8.1
1.7
1.0
70
96
0.013
0.038
beagle
dog
monkey
0.61
2.2
7.7
6.1
1.7
101
0.054
a
2 mg/kg iv dose as a solution in DMSO (rat), 1% Tween80/2%
HMPC/97% water at pH = 4 (dog), or 25% HBC/75% water at pH =
b
4 (cynomolgus monkey). 5 mg/kg oral dose as a solution in 1%
Tween80/2% HMPC/97% water at pH = 2.
cynomolgus monkey was roughly double that of rat and dog, an
observation which can be partially accounted for by the larger
unbound fraction in plasma for monkey relative to rat and dog.
When dosed orally, bioavailability was high (70−101%) across
all three species, and the compound was rapidly absorbed as
indicated by tmax of 1−2 h.
Because 42 achieved good exposure in rats following oral
administration, a dose−response relationship of this compound
on CNS Aβ levels in the rat pharmacodynamic model was
determined. As shown in Figure 3, 42 demonstrated dose-
dependent decreases in both CSF and brain Aβ levels at 4 h
time points. The reduction in Aβ levels tended to be more
significant in the CSF than in the brain, although the values
were generally within 2-fold and ≥70% Aβ reduction was
achieved in both the CSF and brain at a dose of 30 mg/kg. The
effective unbound plasma drug concentration corresponding to
50% Aβ reduction (EC50,unbound) at the 4 h time point was
calculated to be 18 and 67 nM for CSF and brain, respectively.
The temporal effects of 42 on CNS Aβ levels were examined in
CONCLUSION
■
A novel series of potent BACE1 inhibitors was discovered by
incorporating a nitrogen atom into the 4-position of the
aminooxazoline xanthene scaffold. The nitrogen atom led to
improved BACE1 potency, reduced hERG binding affinity, and
elevated Pgp efflux ratios. Optimization of the P3 group and
exploration of the P2′ group resulted in several compounds
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Figure 3. Pharmacodynamic effect of 42 on CNS Aβ in Sprague−Dawley rats. (a) CSF Aβ reduction at 4 h. (b) Brain Aβ reduction at 4 h. (c) Time-
course study at 30 mg/kg.
Figure 4. Comparison of BACE Inhibitors 1 and 42.
Table 6. Aspartyl Protease Selectivity of 1 and 42
protease IC50 (μM)/selectivity
BACE1
BACE2
renin
pepsin
cathepsin D
cathepsin E
1
0.0023
0.0007
0.015 (7×)
0.005 (7×)
>3 (1300×)
>1 (1429×)
9.5 (4130×)
>1 (1429×)
1.1 (478×)
2.3 (3285×)
0.32 (139×)
>1 (1429×)
42
with improved in vitro profiles. An oxetane-containing P2′
group was designed to balance Pgp efflux and microsomal
turnover, and this effort culminated in the identification of
inhibitor 42. Although this compound was a moderate Pgp
substrate, it led to robust and sustained reductions of CSF and
brain Aβ in a rat pharmacodynamic model. In accordance with
low hERG binding affinity, 42 did not result in elongation of
the QTc interval at plasma concentrations exceeding 108-fold
and 31-fold over the plasma EC50,unbound for CSF Aβ reduction
and the vivo plasma IC50,unbound, respectively. Further studies
detailing pharmacodynamic and toxicology studies of this
compound will be reported in due course.
EXPERIMENTAL SECTION
■
BACE1 Enzymatic Assay. BACE1 enzymatic activity was
determined by the enhancement of fluorescence intensity upon
enzymatic cleavage of the fluorescence resonance energy transfer
substrate according to a previously published procedure.6p
Cell-Based Assay. BACE 1 cellular activity was determined in
human embryonic kidney cells (HEK293) stably expressing APPSW
.
After incubation overnight with the test compounds, the conditioned
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media was collected and the Aβ40 levels were determined using a
sandwich ELISA as described previously.6p
Permeability Assay. Permeability and efflux ratios were
determined in the wild-type cell line LLC-PK1 (porcine renal
epithelial cells, WT-LLC-PK1) transfected with human MDR1 gene
(hMDR1-LLC-PK1) and rat mdr1a gene (rMdr1a-LLC-PK1) as
described previously.6p
ice. Acetic acid (16.73 mL, 292 mmol) was added, and the resulting
taffy was stirred vigorously for 2 h, resulting in the formation of a fine
off-white precipitate. The slurry was filtered, and the collected solid
was washed with methanol, washed ether, and then dried to give 2-(4-
bromophenoxy)-5-methoxynicotinic acid (10.5 g, 55% yield) as an off-
1
white solid. H NMR (400 MHz, DMSO-d6) δ = 13.42 (br s, 1 H),
8.06 (d, J = 3.1 Hz, 1 H), 7.81 (d, J = 3.1 Hz, 1 H), 7.60−7.50 (m, 2
H), 7.01−6.94 (m, 2 H), 3.85 (s, 3 H). MS m/z = 324.0 [M + H]+.
Calcd for C13H11BrNO4: 324.0.
Microsomal Stability Assay. Compounds (1 μM) were incubated
with liver microsomes from human and rat for 30 min at 37 °C, and
the samples were analyzed according to published procedure.8
hERG Binding Assay. A stable HEK293 cell line expressing the
hERG channel was established in house. Compounds were tested in
the [3H]-dofetilide binding assay with cell membranes prepared from
this cell line by using the method of Finlayson with some
modifications.22 Briefly, filtration assays were carried out in 194 μL
of binding buffer (10 mM HEPES, pH 7.4, 60 mM KCl, 71.5 mM
NaCl, 1 mM CaCl2, 2 mM MgCl2) with 10 μg/well membrane (based
on membrane protein) and [3H]-dofetilide (8 nM), 6 μL of
compound dissolved in 100% DMSO. Nonspecific binding was
determined by using 10 μM cold dofetilide (∼1000-fold molar excess
over hot ligand). The entire assay was conducted in 96-well Whatman
Unifilter plates at room temperature for 90 min. The binding assay was
terminated by washing the plates four times on a Millipore vacuum
filtration manifold with 100 μL/well of ice-cold wash buffer (10 mM
HEPES, pH 7.4, 131.5 mM NaCl, 1 mM CaCl2, 2 mM MgCl2). The
bound radioisotope was quantified using a Packard TopCount NTS
liquid scintillation counter with scintillation fluid.
Step 2: 7-Bromo-3-methoxy-5H-chromeno[2,3-b]pyridin-5-one. 2-
(4-Bromophenoxy)-5-methoxynicotinic acid (8.00 g, 24.68 mmol) was
added to a round-bottom flask containing polyphosphoric acid (115%
H3PO4) (75 g, 24.68 mmol) that had been warmed to 140 °C. The
resulting mixture was stirred at this temperature for 3.5 h. The reaction
mixture was allowed to cool to 100 °C and then poured into 500 mL
of ice−water. The resulting thick taffy was stirred vigorously, leading to
the formation of a fine white precipitate. Stirring was continued for 1
h, at which point the mixture was filtered. The collected solid was
washed with MeOH (2 × 100 mL), washed with diethyl ether (3 ×
100 mL), and dried to give 7-bromo-3-methoxy-5H-chromeno[2,3-
1
b]pyridin-5-one (7.05 g, 93% yield) as a fine-white solid. H NMR
(400 MHz, DMSO-d6) δ = 8.71 (d, J = 3.3 Hz, 1 H), 8.35 (d, J = 2.4
Hz, 1 H), 8.20−8.12 (m, 2 H), 7.86 (d, J = 8.9 Hz, 1 H), 4.07 (s, 3 H).
(S)-7-Bromo-3-methoxy-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-2′-amine (6). To a slurry of 7-bromo-3-methoxy-5H-
chromeno[2,3-b]pyridin-5-one (23 g, 75 mmol) in THF (751 mL,
75 mmol) at −40 °C was added methylmagnesium chloride (3 M
solution in THF, 88 mL, 263 mmol) over 2 min, such that the
temperature did not rise above −35 °C. The resulting red slurry was
maintained at −30 °C. After 1 h, the reaction mixture was quenched
by the addition of EtOAc (50 mL). The solution was then carefully
quenched with 800 mL of a 50% aq ammonium chloride solution. The
mixture was poured into a separatory funnel containing ethyl acetate
(100 mL). The layers were separated, and the organic layer was
washed with brine, dried over sodium sulfate, filtered, and
concentrated. The aqueous layer was extracted with ethyl acetate (3
× 500 mL). The combined organic layers were washed with water and
then brine, dried over sodium sulfate, filtered, and combined with the
first lot of residue. This organic solution was washed with brined, dried
over sodium sulfate, fitlered, and concentrated to provide a yellow
solid which was used directly in the next reaction.
Pharmacodynamic Assay. Male Sprague−Dawley rats (175−200
g) were administered compound by oral gavage at the appropriate
dose. Samples of plasma, CSF, and brain were analyzed according to
previously reported procedures.6p
Chemistry. Unless otherwise noted, all materials were obtained
from commercial suppliers and used without further purification.
Anhydrous solvents were obtained from Aldrich or EM Science and
used directly. All reactions involving air- or moisture-sensitive reagents
were performed under a nitrogen or argon atmosphere. All microwave
assisted reactions were conducted with a Smith synthesizer from
Personal Chemistry, Uppsala, Sweden. Silica gel chromatography was
performed using either glass columns packed with silica gel (230−400
mesh, EMD Chemicals, Gibbstown, NJ) or prepacked silica gel
1
cartridges (Biotage or ISCO). H NMR spectra were recorded on a
Step 2: To a solution of 7-bromo-3-methoxy-5-methyl-5H-
chromeno[2,3-b]pyridin-5-ol (23.5 g, 72.9 mmol, from step 1) in
THF (729 mL, 72.9 mmol) was added HCl (1 M in ether) (0.729 mL,
0.729 mmol). After heating at 45 °C for 10 min, more HCl (1 M in
ether) (0.729 mL, 0.729 mmol) was added. The reaction was heated at
45 °C for a total of 30 min, at which point TLC indicated complete
conversion to olefin. This light-yellow solution was cooled to −25 °C
and added to the slurry generated below.
Bruker AV-400 (400 MHz) spectrometer at ambient temperature or
on a Varian 400 MHz spectrometer. Chemical shifts are reported in
parts per million (ppm, δ units) downfield from tetramethylsilane.
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. Purity for final compounds was
greater than 95% unless otherwise noted and was measured using
Agilent 1100 series high performance liquid chromatography (HPLC)
systems with UV detection at 254 nm (system A, Agilent Zorbax
Eclipse XDB-C8 4.6 mm × 150 mm, 5 μm, 5−100% CH3CN in H2O
with 0.1% TFA for 15 min at 1.5 mL/min; system B, Waters Xterra 4.6
mm × 150 mm, 3.5 μm, 5−95% CH3CN in H2O with 0.1% TFA for
15 min at 1.0 mL/min). Exact mass confirmation was performed on an
Agilent 1100 series high performance liquid chromatography (HPLC)
system (Santa Clara, CA, U.S.) by flow injection analysis, eluting with
a binary solvent system A and B (A, water with 0.1% FA; B, ACN with
0.1% FA) under isocratic conditions (50% A/50% B) at 0.2 mL/min
with MS detection by an Agilent G1969A time-of-flight (TOF) mass
spectrometer (Santa Clara, CA, U.S.).
7-Bromo-3-methoxy-5H-chromeno[2,3-b]pyridin-5-one (5). Step
1: 2-(4-Bromophenoxy)-5-methoxynicotinic acid. To a slurry of
sodium hydride (60% dispersion) (5.14 g, 129 mmol) in DMF (106
mL, 58.4 mmol) at 0 °C was added 4-bromophenol (11.12 g, 64.3
mmol), resulting in the evolution of hydrogen gas. The mixture was
stirred at 0 °C for 2 min then for an additional 5 min at room
temperature. 2-Fluoro-5-methoxynicotinic acid (10.0 g, 58.4 mmol)
was added portionwise over 1 min. The resulting slurry was heated to
130 °C for 20 h, then heated to 140 °C for another 3 h. The reaction
mixture was cooled to room temperature and poured onto 500 g of
To a solution of iodine (20.37 g, 80 mmol) and THF (400 mL) at
−15 °C was added silver cyanate (32.8 g, 219 mmol). The resulting
slurry was maintained at −40 °C for 25 min, and then the olefin
solution (generated above) was added via cannula over 15 min,
maintaining the temperature below −35 °C. The derived slurry was
maintained at −30 °C for 1 h and then was filtered through a pad of
Celite, washing well with 200 mL of THF. The derived brown solution
was cooled to −20 °C and treated with ammonia (2.0 M in 2-
propanol, 219 mL, 438 mmol). The resulting solution was allowed to
slowly warm to room temperature overnight. To the reaction was
added 10% aq sodium thiosulfate solution (700 mL), and the resulting
light-orange solution was stirred for 10 min before being poured into a
separatory funnel containing EtOAc (250 mL). The layers were
separated, and the organic layer was washed with brine and
concentrated in vacuo to provide an orange mixture still containing
a considerable amount of water. This mixture was combined with the
organic extracts obtained below.The aqueous layer was extracted with
ethyl acetate (2 × 500 mL). These organics were combined with the
organics obtained and poured into a separatory funnel. The layers were
separated, and the aqueous layer was extracted with ethyl acetate (2 ×
100 mL). The combined organic layers were washed with brine, dried
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over sodium sulfate, filtered, and concentrated in vacuo to provide 25 g
of racemic 7-bromo-3-methoxy-5′H-spiro[chromeno[2,3-b]pyridine-
5,4′-oxazol]-2′-amine as a brown solid. This material was dissolved in
DCM (200 mL) and MeOH (200 mL). The solution was purified by
chromatography, using 6 mL injections on a 5 μm, 5 cm × 15 cm
Chiralpak AD column with 50% MeOH/0.2% diethylamine/50% CO2
at a flow rate of 250 mL/min to afford peak 1, (R)-7-bromo-3-
methoxy-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine
(8.235 g, 22.74 mmol, 31.2% yield), and peak 2, (S)-7-bromo-3-
methoxy-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine
(7.53 g, 20.79 mmol, 28.5% yield). 1H NMR (400 MHz, DMSO-d6) δ
= 8.30−8.22 (m, 1 H), 7.66 (d, J = 2.2 Hz, 1 H), 7.17 (d, J = 9.0 Hz, 1
H), 6.96 (dd, J = 3.0, 8.9 Hz, 1 H), 6.83 (d, J = 3.0 Hz, 1 H), 6.55 (br
s, 2 H), 5.50 (s, 1 H), 4.29−4.10 (m, 2 H), 3.76 (s, 3 H). MS m/z =
362.0 [M + H]+. Calcd for C15H13BrN3O: 362.0.
with 4-iodophenol (58.6 g, 266 mmol) portionwise, resulting in
bubbling. After the bubbling had subsided, 5-bromo-2-chloronicotinic
acid (60.2 g, 255 mmol) was then added to slurry portionwise and the
resulting mixture was heated to 115 °C overnight. The mixture was
cooled to room temperature then diluted with water (240 mL). Acetic
acid (ca. 60 mL) was added until the mixture achieved pH of 4−5.
Water (1260 mL) was added via addition funnel, and the resulting
mixture was cooled in an ice-bath for 1.5 h. The mixture was filtered,
and the collected solid was washed with water and dried on the filter
overnight to give 5-bromo-2-(4-iodophenoxy)nicotinic acid (80.9 g,
1
76%) as a light-brown solid. H NMR (400 MHz, DMSO-d6) δ =
13.56 (br s, 1 H), 8.40 (dd, J = 2.6, 13.4 Hz, 2 H), 7.82−7.60 (m, 2
H), 7.05−6.80 (m, 2 H). MS m/z = 420.0 [M + H]+. Calcd for
C12H8BrINO3: 419.9.
Step 2: 3-Bromo-7-iodo-5H-chromeno[2,3-b]pyridin-5-one, 5-
bromo-2-(4-iodophenoxy)nicotinic acid (80.9 g, 193 mmol) was
added portionwise to a round-bottom flask containing polyphosphoric
acid at 110 °C. When the addition was complete, the mixture was
stirred at 110 °C overnight and then at 140 °C for 1.5 h. The mixture
was cooled to 35 °C and then added slowly to ice-cold water (1.2 L).
The resulting slurry was stirred for 1 h at room temperature, and then
the solid was filtered. The collected solid was washed with water (2×),
washed with 2-PrOH, and dried under vacuum to give 3-bromo-7-
iodo-5H-chromeno[2,3-b]pyridin-5-one (65.9 g, 85%) as a white solid.
1H NMR (400 MHz, DMSO-d6) δ = 8.95 (d, J = 2.6 Hz, 1 H), 8.71 (d,
(S)-2′-Amino-7-bromo-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-3-ol (7). To a solution of (S)-7-bromo-3-methoxy-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (6) (6.0 g,
16.57 mmol) in DCM (166 mL) at 0 °C was added boron tribromide
(9.40 mL, 99 mmol). This led to the immediate formation of a light-
orange precipitate. The resulting slurry was removed from the ice bath
and allowed to stir at room temperature for 12 h. The mixture was
cooled to −20 °C and treated with methanol (6.70 mL, 166 mmol),
followed immediately by saturated aq sodium bicarbonate (1 L). DCM
(500 mL) was added, and the mixture was warmed to room
temperature. The layers were separated, and the aqueous layer was
extracted with methylene chloride (500 mL). The combined organic
layers were washed with brine and dried over sodium sulfate. The
aqueous layer was then extracted with ethyl acetate (2 × 500 mL). The
combined organics were washed with brine, dried over sodium sulfate,
and combined with the organic solution obtained above. The aqueous
layer was then stirred overnight with 500 mL of ethyl acetate. The
organic layer was removed, washed with brine, dried over sodium
sulfate, and filtered. This solution was combined with the organic
layers above and concentrated in vacuo to provide an orange solid.
This solid was taken up in 10 mL of DCM and purified by silica gel
chromatography (0−10% MeOH/DCM with 0.1% ammonium
hydroxide) to provide (S)-2′-amino-7-bromo-5′H-spiro[chromeno-
[2,3-b]pyridine-5,4′-oxazol]-3-ol (2.95 g, 8.47 mmol, 51.1% yield) as a
J = 2.5 Hz, 1 H), 8.39 (d, J = 2.2 Hz, 1 H), 8.20 (dd, J = 2.2, 8.8 Hz, 1
H), 7.59 (d, J = 8.8 Hz, 1 H).
(S)-3-Bromo-7-iodo-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxa-
zol]-2′-amine (12). Step 1: A mixture of 3-bromo-7-iodo-5H-
chromeno[2,3-b]pyridin-5-one (33.0 g, 82 mmol) and THF (1642
mL, 82 mmol) in a 3 L three-neck flask was cooled to −40 °C. To this
slurry was added methylmagnesium bromide (1 M in butyl ether)
(164 mL, 164 mmol) via an addition funnel over 10 min. The internal
temperature rose to −30 °C during the addition. The flask was
maintained in the cooling bath and slowly allowed to warm to 0 °C
over the course of 2 h. The reaction mixture was diluted with saturated
aq ammonium chloride (1 L) and poured into a separatory funnel
containing ethyl acetate (250 mL). The layers were separated, and the
aqueous layer was extracted with ethyl acetate (2 × 250 mL). The
combined organic layers were washed with brine, dried over sodium
sulfate, filtered, and concentrated in vacuo (maintained on the rotovap
at 80 °C for 30 min) to provide the tertiary alcohol as a light-yellow
solid. The derived alcohol was taken up in 150 mL of THF and 50 mL
of chloroform and maintained at 80 °C for 30 min. The yellow solid
was concentrated under reduced pressure to give the crude olefin as an
orange solid. The material was used directly in next step.
Step 2: A 2 L round-bottom flask charged with iodine (19.99 g, 79
mmol) and 600 mL of THF was cooled in a dry ice acetone bath until
an internal temperature of −25 °C was maintained, at which point
silver cyanate (33.7 g, 225 mmol) was added in one portion. During
this addition, the internal temperature rose to −20 °C. The resulting
slurry was maintained at between −25 and −20 °C for 20 min, at
which point a solution of the crude 3-bromo-7-iodo-5-methylene-5H-
chromeno[2,3-b]pyridine (30.0 g, 75.0 mmol) in 500 mL of THF was
added via cannula over the course of 5 min. The internal temperature
rose to −10 °C during this addition. The flask was recooled to −20 °C
and maintained between −20 and −10 °C for 1.5 h. The reaction
mixture was filtered through a plug of Celite (500 g) with the aid of
diethyl ether (until the filtrate was colorless). The filtrate was
concentrated in vacuo, and the resulting brown oily residue was taken
up in THF (1 L). The resulting mixture was cooled to 0 °C and
treated with ammonia (2 M in 2-propanol, 225 mL, 450 mmol).
Following the addition, the flask was removed from the ice bath and
maintained stirred for 8 h. The reaction mixture was diluted with 10%
aq sodium thiosulfate solution (1 L) and poured into a separatory
funnel containing ethyl acetate (500 mL) and brine (250 mL). The
layers were separated, and the aqueous layer was extracted with ethyl
acetate (2 × 500 mL). The combined organic layers were washed with
brine, dried over sodium sulfate, filtered, and concentrated in vacuo to
provide a brown residue. The residue was dried further by
1
light-orange solid. H NMR (400 MHz, DMSO-d6) δ = 9.88 (br s, 1
H), 7.81 (d, J = 2.7 Hz, 1 H), 7.59−7.44 (m, 2 H), 7.28−7.12 (m, 2
H), 4.31 (br s, 2 H), 4.06 (q, J = 5.2 Hz, 1 H), 3.18 (s, 1 H), 3.16 (s, 1
H).
(S)-7-Bromo-3-(neopentyloxy)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-2′-amine (8). To a solution of (S)-2′-amino-7-
bromo-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-3-ol (0.900 g,
2.59 mmol) in N,N-dimethylformamide (10.34 mL, 2.59 mmol) at rt
was added cesium carbonate (2.106 g, 6.46 mmol). The mixture was
stirred for a 1 min, and then 1-iodo-2,2-dimethylpropane (0.514 mL,
3.88 mmol) was added. The mixture was heated to 100 °C for 3 h.
After cooling to room temperature, the reaction mixture was diluted
with water (250 mL) and poured into a separatory funnel containing
ethyl acetate (250 mL). The layers were separated, and the aqueous
layer was extracted with ethyl acetate (3 × 200 mL). The combined
organic layers were washed with water and then brine, dried over
sodium sulfate, filtered, and concentrated. The aq layer was then
extracted with DCM (4 × 150 mL). The combined organics were
washed with brine, dried over sodium sulfate, filtered, and
concentrated. The two residues were combined, and the combined
residue was purified by chromatography on silica gel (0−10% MeOH/
DCM 0.1% ammonium hydroxide). The resulting material was
purified again by chromatography on silica gel (0−100% EtOAc/
DCM) to provide (S)-7-bromo-3-(neopentyloxy)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (680 mg, 1.626
mmol, 62.9% yield) as an orange solid. MS m/z = 418.0 [M + H]+.
Calcd for C19H21BrN3O3: 418.0.
3-Bromo-7-iodo-5H-chromeno[2,3-b]pyridin-5-one (11). Step 1:
5-Bromo-2-(4-iodophenoxy)nicotinic acid, a round-bottom flask under
N2(g) was charged with sodium hydride (22.40 g, 560 mmol) and
DMF (540 mL). The slurry was cooled to 0 °C and carefully treated
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concentration from toluene (3 × 100 mL). The residue was then
triturated with DCM/MeOH (600 mL/300 mL) and cooled to 0 °C
overnight. The mixture was then filtered. The solid was collected and
the filtrate was concentrated, and the trituration procedure was
repeated twice more. The collected solids were combined, and the
filtrate was further purified by chromatography on silica gel (0−10%
MeOH/DCM with 0.1% ammonium hydroxide). The product thus
obtained was combined with solid to give 21.9 g of racemic (S)-3-
bromo-7-iodo-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-
amine. A portion of this material (16 g) was dissolved in a mixture of
DCM (150 mL), MeOH (150 mL), and DMSO (25 mL). The
solution was purified by SFC, using 6 mL injections on a 5 μm, 5 cm ×
15 cm Chiralpak AD column with 35% MeOH/0.2% diethylamine/
65% CO2 at a flow rate of 250 mL/min. This afforded peak 1, (R)-3-
bromo-7-iodo-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-
amine (4.50 g, 9.82 mmol, 13.10% yield), and peak 2, (S)-3-bromo-7-
iodo-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (4.45
g, 9.72 mmol, 12.95% yield). 1H NMR (400 MHz, DMSO-d6) δ
ppm 8.40 (d, J = 2.55 Hz, 1 H), 7.87 (d, J = 2.54 Hz, 1 H), 7.70 (dd, J
= 8.56, 2.20 Hz, 1 H), 7.58 (d, J = 2.05 Hz, 1 H), 7.08 (d, J = 8.51 Hz,
1 H), 6.63 (s, 2 H), 4.16−4.30 (m, 2 H).
(S)-3-Bromo-7-(2-fluoropyridin-3-yl)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-2′-amine (13). A 50 mL round-bottom flask
was charged with (S)-3-bromo-7-iodo-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-2′-amine (2.56 g, 5.59 mmol), 2-fluoro-3-
pyridineboronic acid (1.103 g, 7.82 mmol), potassium carbonate
(2.317 g, 16.77 mmol), and 1,1′-bis(diphenylphosphino)ferrocene-
palladium dichloride (0.228 g, 0.279 mmol). The flask was flushed
with Ar(g), and then 1,4-dioxane (11.18 mL) and water (5.5 mL) were
added in sequence. The flask was fitted with a reflux condenser and
heated to 70 °C for 40 min. The mixture was cooled to room
temperature and diluted with EtOAc (20 mL) and water (20 mL). The
layers were separated, and the aqueous layer was extracted with EtOAc
(20 mL). The organic layers were combined, and the combined
solution was dried over sodium sulfate, filtered, and evaporated. The
residue was purified by chromatography on silica gel (0−100%
EtOAc/heptane) to give (S)-3-bromo-7-(2-fluoropyridin-3-yl)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (2.172 g, 5.08
mmol, 91% yield) as an orange solid. 1H NMR (400 MHz, DMSO-d6)
δ = 8.42 (d, J = 2.4 Hz, 1 H), 8.29−8.21 (m, 1 H), 8.12 (ddd, J = 2.0,
7.4, 10.4 Hz, 1 H), 7.88 (d, J = 2.4 Hz, 1 H), 7.67−7.61 (m, 1 H), 7.57
(t, J = 1.8 Hz, 1 H), 7.49 (ddd, J = 2.0, 4.9, 7.5 Hz, 1 H), 7.38 (d, J =
8.6 Hz, 1 H), 6.58 (s, 2 H), 4.36−4.21 (m, 2 H). MS m/z = 427.0 [M
+ H]+. Calcd for C19H13BrFN4O2: 427.0.
The partially cooled reaction mixture was poured onto 750 g of ice.
The resulting mixture was then carefully adjusted with 10 M aq NaOH
solution to a pH of 9−10 (during this quench the internal temperature
rose to 100 °C). The mixture was cooled to room temperature and
filtered. This material was taken up in DCM (200 mL), MeOH (200
mL), and benzene (100 mL). The mixture was concentrated in vacuo.
The residue was taken up in DCM (400 mL) and MeOH (400 mL),
and the resulting orange slurry was heated to 65 °C for 1 h, and then
the hot solution was filtered. The filtrate was concentrated to afford 3-
bromo-7-methoxy-5H-chromeno[2,3-b]pyridin-5-one (53.3 g, 174
mmol, 66.4% yield) as an orange solid. 1H NMR (400 MHz,
DMSO-d6) δ = 8.94 (d, J = 2.6 Hz, 1 H), 8.72 (d, J = 2.6 Hz, 1 H),
7.77−7.70 (m, 1 H), 7.61−7.50 (m, 2 H), 3.90 (s, 3 H). MS m/z =
306.0 [M + H]+. Calcd for C13H9BrNO3: 306.0.
(S)-3-Bromo-7-methoxy-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-2′-amine (16). Step 1: A three-neck 5 L flask equipped with
an addition funnel and overhead stirrer was charged with 3-bromo-7-
methoxy-5H-chromeno[2,3-b]pyridin-5-one (52.00 g, 170 mmol).
THF (1.7 L) was added, and the reaction vessel was cooled in an
ice bath. When the internal temperature reached 5 °C, methyl-
magnesium bromide (1 M in butyl ether) (340 mL, 340 mmol) was
added dropwise via an addition funnel over the course of 15 min. The
red slurry was removed from the ice bath and stirred at rt for 1.5 h.
The reaction was carefully quenched with saturated aq ammonium
chloride solution (1.2 L). DCM (1 L) was added, and the yellow
mixture was stirred for 30 min. The layers were separated, and the
aqueous layer was extracted with DCM (3 × 500 mL). The organic
layers were each individually washed with brine and then combined.
The combined solution was dried over sodium sulfate, filtered, and
concentrated in vacuo at 75 °C provide an orange solid. The solid was
suspended in MeOH (700 mL) and heated to 60 °C. The warm slurry
was filtered to give 21.5 g of a light-yellow solid. The filtrate was
concentrated, and the residue was purified by chromatgraphy on silica
gel (eluting with DCM) to afford another 6.0 g of solid. The two solids
were combined and dried under vacuum to give 3-bromo-7-methoxy-
5-methylene-5H-chromeno[2,3-b]pyridine (27.5 g, 53% yield) as a
light-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ = 8.60 (d, J = 2.3
Hz, 1 H), 8.38 (d, J = 2.4 Hz, 1 H), 7.34 (d, J = 2.9 Hz, 1 H), 7.20 (d, J
= 9.0 Hz, 1 H), 7.02 (dd, J = 2.9, 9.0 Hz, 1 H), 5.95 (d, J = 1.1 Hz, 1
H), 5.90 (d, J = 1.0 Hz, 1 H), 3.82 (s, 3 H),
Step 2: A round-bottom flask was charged with iodine (18.49 g, 72.8
mmol) and THF (347 mL, 69.4 mmol). The resulting solution was
cooled to −15 °C and treated in one portion with silver isocyanate
(31.2 g, 208 mmol). The temperature, which rose to −12 °C during
the addition, was lowered to −20 °C and maintained for 15 min. A
solution of 3-bromo-7-methoxy-5-methylene-5H-chromeno[2,3-b]-
pyridine (21.10 g, 69.4 mmol) in THF (347 mL, 69.4 mmol) was
added, and the resulting mixture was maintained at −20 to −10 °C for
1 h. The yellow slurry was diluted with ether (250 mL) and filtered
through a pad of celilte, washing well with ether and THF. The
resulting brown filtrate was concentrated under reduced pressure, with
minimal heating. The derived brown oil was taken up in THF (347
mL, 69.4 mmol), cooled to 0 °C, and treated with ammonia (2 M in 2-
PrOH) (104 mL, 208 mmol). The solution was maintained at 0 °C for
15 min then warmed to room temperature, where it was maintained
for 10 h. The reaction was diluted with 10% aq sodium thiosulfate
solution (1 L) and poured into a separatory funnel containing EtOAc
(250 mL). The layers were separated, and the aq layer was extracted
with EtOAc (3 × 250 mL). The combined organic extracts were
washed with brine, dried over sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified by silical gel
chromatography (0−100% EtOAc/hexanes) to provide rac-3-bromo-
7-methoxy-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine
(17.5 g, 48.3 mmol, 69.6% yield) as a yellow solid. This material was
dissolved in 1:1 DCM/MeOH (300 mL), and the resulting solution
was purified by SFC, using 4 mL injections on a 5 cm × 25 cm
Chiralpak AD-H column with 35% MeOH/0.2% diethylamine/65%
CO2 at a flow rate of 250 mL/min to give peak 1, (R)-3-bromo-7-
methoxy-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine
(6.47 g), and peak 2, (S)-3-bromo-7-methoxy-5′H-spiro[chromeno-
3-Bromo-7-methoxy-5H-chromeno[2,3-b]pyridin-5-one (15).
Step 1: To a round-bottom flask charged with sodium hydride (60%
dispersion in oil, 26.6 g, 666 mmol) was added N,N-dimethylforma-
mide (634 mL, 317 mmol). The slurry was cooled to 0 °C and
carefully treated with 4-methoxyphenol (39.4 g, 317 mmol)
portionwise over 7 min, resulting in the evolution of a large amount
of hydrogen gas. The mixture was stirred for 2 min, and then the ice
bath was removed. After another 5 min of stirring, 5-bromo-2-
chloronicotinic acid (75 g, 317 mmol) was slowly added portionwise
over 5 min. After the addition was complete, the solution was heated
to 140 °C for 1 h. The reaction mixture was cooled to room
temperature and diluted with ice−water (750 mL). The brown
mixture was acidified with acetic acid (91 mL, 1586 mmol), and then
concentrate hydrochloric acid (93 mL, 1110 mmol) was added,
resulting in the formation of a white precipitate. The pink slurry was
stirred at room temperature for 2 h before being filtered to provide an
off-white solid. This white solid contained residual DMF and was
further dried under vacuum at 80 °C for 1 h to provide 5-bromo-2-(4-
methoxyphenoxy)nicotinic acid (85.10 g, 263 mmol, 83% yield) as a
1
white solid. H NMR (400 MHz, chloroform-d) δ = 8.58 (d, J = 2.5
Hz, 1 H), 8.35 (d, J = 2.6 Hz, 1 H), 7.16−7.06 (m, 2 H), 7.02−6.92
(m, 2 H), 3.84 (s, 3 H).
Step 2: 5-Bromo-2-(4-methoxyphenoxy)nicotinic acid (85 g, 262
mmol) was added to a round-bottom flask containing polyphosphoric
acid (900 g, 262 mmol) which had been heated to 140 °C. The
mixture was maintained at 140 °C for 2 h and then cooled for 15 min.
L
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[2,3-b]pyridine-5,4′-oxazol]-2′-amine (7.12 g, 28% yield). Data for
peak 2: 1H NMR (400 MHz, DMSO-d6) δ = 8.37 (d, J = 2.5 Hz, 1 H),
7.83 (d, J = 2.4 Hz, 1 H), 7.18 (d, J = 8.9 Hz, 1 H), 6.96 (dd, J = 3.1,
8.9 Hz, 1 H), 6.81 (d, J = 2.9 Hz, 1 H), 6.58 (br s, 2 H), 4.32−4.12 (m,
2 H), 3.76 (s, 3 H).
(tetrahydro-2H-pyran-4-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-7-ol (201.8 mg, 0.698 mmol, 82% yield) as an off-white solid.
1H NMR (400 MHz, DMSO-d6) δ = 9.31 (s, 1 H), 8.13 (d, J = 2.3 Hz,
1 H), 7.60 (d, J = 2.4 Hz, 1 H), 7.09−6.97 (m, 1 H), 6.81−6.68 (m, 2
H), 6.48 (s, 2 H), 4.16−4.08 (m, 2 H), 3.95 (dd, J = 1.9, 9.5 Hz, 2 H),
3.48−3.38 (m, 2 H), 2.91−2.78 (m, 1 H), 1.77−1.56 (m, 4 H). MS m/
z = 354.0 [M + H]+. Calcd for C19H20N3O4: 354.2.
(S)-2′-Amino-3-bromo-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-7-ol (17). A 250 mL round-bottom flask was charged with a
solution of (S)-3-bromo-7-methoxy-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-2′-amine (1.832 g, 5.06 mmol) in DCM (50.6
mL). The flask was cooled in an ice-bath for 10 min, and then BBr3
(1.913 mL, 20.23 mmol) was added over 30 s, slightly faster than
dropwise. The mixture was stirred for 5 min, and then the ice-bath was
removed. The mixture was stirred for 5 h at room temperature and
then was diluted with saturated aq sodium bicarbonate solution (75
mL). A solid formed, so THF (30 mL) was added and the mixture was
stirred overnight. The layers were separated, and the aqueous layer was
extracted with DCM (3 × 50 mL). The combined organic extracts
were dried over sodium sulfate, filtered, and evaporated to give an
orange solid. The solid was taken up in DCM (50 mL), and the
resulting mixture was sonicated for 10 min. The mixture was then
cooled in an ice-bath for 20 min. The solid was filtered, washed with
cold DCM (30 mL), and dried under vacuum to give give (S)-2′-
amino-3-bromo-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-7-ol
(1.089 g, 3.13 mmol, 61.8% yield) as a cream-colored solid. MS m/z =
347.8 [M + H]+. Calcd for C14H11BrN3O3: 348.0.
(S)-2′-Amino-3-(6,6-dimethyl-3,6-dihydro-2H-pyran-4-yl)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-7-ol (18b). A microwave
reaction vial was charged with (S)-2′-amino-3-bromo-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-7-ol (0.850 g, 2.441 mmol),
2-(2,2-dimethyl-3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane (as a 1.5:1 mixture of olefin isomers, 0.930 g, 3.91
mmol), potassium carbonate (1.012 g, 7.32 mmol) and
PdCl2(AmPhos)2 (0.086 g, 0.122 mmol), 1,4-dioxane (14.24 mL),
and water (2.035 mL). The vial was sealed and heated in microwave
reactor for 1 h at 100 °C. The reaction mixture was diluted with water
and washed with EtOAc. The aqueous layer was extracted with EtOAc,
and the combined organic layers were dried with sodium sulfate,
filtered, and concentrated. The residue was purified via column
chromatography on silica gel (0−80% of a 90:10:1 mixture of DCM/
MeOH/NH4OH in DCM). The resulting material was repurified to
collect the title regiosomer as the first eluting peak to afford (S)-2′-
amino-3-(6,6-dimethyl-3,6-dihydro-2H-pyran-4-yl)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-7-ol (0.604 g, 1.592 mmol,
1
3-Bromo-7-hydroxy-spiro[chromeno[2,3-b]pyridine]-5,4′-[1,3]-
oxazole]-2′-amine (rac-17). To a solution of rac-3-bromo-7-methoxy-
spiro[chromeno[2,3-b]pyridine]-5,4′-[1,3]oxazole]-2′-amine (2300
mg, 6350 μmol) in DCM (12.7 mL 6.35 mmol) at 0 °C was added
BBr3 (1.8 mL, 19 mmol). Immediately a thick precipitate formed. The
resulting red slurry was stirred at 0 °C for 10 min, at which point the
ice bath was removed and the mixture was allowed to warm to rt and
stirred at rt for 1 h. Another portion of BBr3 (1 mL) at 23 °C, and the
mixture was stirred for 1 h. The reaction was cooled to 0 °C and
carefully quenched with saturated aq sodium bicarbonate solution
(250 mL) and then poured into DCM (250 mL). The layers were
separated, and the aqueous layer was extracted with DCM (3 × 300
mL). The organic layers were washed with brine, dried over Na2SO4,
and filtered. The extraction process was repeated with DCM. All
organic layers were combined and concentrated under reduced
pressure to provide 3-bromo-7-hydroxy-spiro[chromeno[2,3-b]-
pyridine]-5,4′-[1,3]oxazole]-2′-amine (2.2 g, 99% yield) as a brown
solid. MS m/z = 348.0 [M + H]+. Calcd for C14H11BrN3O3: 348.0.
(S)-2′-Amino-3-(tetrahydro-2H-pyran-4-yl)-5′H-spiro[chromeno-
[2,3-b]pyridine-5,4′-oxazol]-7-ol (18a). Step 1: A vial was charged
with (S)-2′-amino-3-bromo-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-7-ol (380 mg, 1.091 mmol), potassium carbonate (754 mg,
5.46 mmol), 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane (688 mg, 3.27 mmol), Pd(Ph3P)4 (126 mg, 0.109
mmol), DMF (5457 μL), and water (2.5 mL). The vial was sealed and
heated to 80 °C overnight. The mixture was diluted with water (35
mL) and extracted with EtOAc (3 × 15 mL). The combined organic
extracts were dried over sodium sulfate, filtered, and evaporated. The
residue was purified by chromatography on silica gel (0−100% of a
90:10:1 mix of DCM/MeOH/NH4OH in DCM) to give give (S)-2′-
amino-3-(3,6-dihydro-2H-pyran-4-yl)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-7-ol (267.7 mg, 0.762 mmol, 69.8% yield) as an
orange solid. MS m/z = 352.0 [M + H]+. Calcd for C19H18N3O4:
352.1.
65.2% yield) as a tan solid. H NMR (400 MHz, DMSO-d6) δ = 9.35
(br s, 1 H), 8.31 (d, J = 2.4 Hz, 1 H), 7.73 (d, J = 2.4 Hz, 1 H), 7.08−
7.00 (m, 1 H), 6.83−6.69 (m, 2 H), 6.50 (s, 2 H), 6.17 (t, J = 1.5 Hz, 1
H), 4.14 (s, 2 H), 3.83 (t, J = 5.4 Hz, 2 H), 2.40−2.32 (m, 2 H), 1.27
(s, 3 H), 1.27 (s, 3 H). MS m/z = 380.2 [M + H]+. Calcd for
C21H22N3O4: 380.2.
(S)-2′-Amino-3-morpholino-5′H-spiro[chromeno[2,3-b]pyridine-
5,4′-oxazol]-7-ol (18c). A vial was charged with (S)-2′-amino-3-
bromo-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-7-ol (0.647 g,
1.858 mmol), DavePhos (0.088 g, 0.223 mmol), and tris-
(dibenzylideneacetone)dipalladium(0) (0.085 g, 0.093 mmol). The
vessel was flushed with Ar(g), and then lithium bis(trimethylsilyl)-
amide (1.0 M in THF) (9.29 mL, 9.29 mmol) and morpholine (0.486
mL, 5.58 mmol) were added in sequence. The vial was sealed and
heated at 70 °C for 1 h. The mixture was diluted with water and
saturated aq ammoniom chloride solution. The mixture was extracted
with DCM (3 × 30 mL) and EtOAc (3×). The combined organic
extracts were combined, dried over sodium sulfate, filtered, and
evaporated. The residue was purified by chromatography on silica gel
(0−10% of a 90:10:1 mix of DCM/MeOH/NH4OH in DCM) to
afford (S)-2′-amino-3-morpholino-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-7-ol (0.336 g, 0.948 mmol, 51.0% yield) as an
orange solid. MS m/z = 355.2 [M + H]+. Calcd for C18H19N4O4:
355.1.
(S)-2′-Amino-3-(p-tolyl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-7-ol (18d). A vial was charged (S)-2′-amino-3-bromo-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-7-ol (282 mg, 0.809
mmol), p-tolylboronic acid (220 mg, 1.618 mmol), potassium
carbonate (559 mg, 4.04 mmol), and Pd(Ph3P)4 (46.7 mg, 0.040
mmol). The vial was flushed with Ar(g), and then 1,4-dioxane (4 mL)
and water (2 mL) were added in sequence. The vial was sealed and
heated to 80 °C for 1.5 h. The mixture was partitioned between brine
and 10% iPrOH/EtOAc. The layers were separated, and the aqueous
layer was extracted with EtOAc. The combined organic extracts were
dried over sodium sulfate, filtered, and evaporated. The residue was
purified by chromatography on silica gel (0−80% of a 90:10:1 mix of
DCM/MeOH/NH4OH in DCM) to give (S)-2′-amino-3-p-tolyl-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-7-ol (259.36 mg, 0.722
mmol, 89% yield) as an orange solid. MS m/z = 360.0 [M + H]+.
Calcd for C21H18N3O3: 360.1.
Step 2: A 25 mL flask was charged with (S)-2′-amino-3-(3,6-
dihydro-2H-pyran-4-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxa-
zol]-7-ol (211 mg, 0.601 mmol) and MeOH (7.5 mL). The mixture
was sonicated for 1 min to give an opaque mixture. 10% Pd/C (63.9
mg, 0.060 mmol) was added, and H2(g) was bubbled through the
mixture for 1 min. The mixture was stirred further under a balloon of
H2(g) for 24 h. H2(g) was again bubbled in for 1 min, and the mixture
was stirred for 2 days under a H2(g) balloon. The mixture was filtered
through Celite with the aid of methanol. The filtrate was evaporated,
and the residue was purified by chromatography on silica gel (0−100%
of a 90:10:1 mix of DCM/MeOH/NH4OH) to give (S)-2′-amino-3-
(S)-4-(2′-Amino-7-hydroxy-5′H-spiro[chromeno[2,3-b]pyridine-
5,4′-oxazol]-3-yl)benzonitrile (18e). A vial was charged with (S)-2′-
amino-3-bromo-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-7-ol
(247 mg, 0.711 mmol), 4-cyanophenylboronic acid (313 mg, 2.132
mmol), potassium carbonate (491 mg, 3.55 mmol), and Pd(Ph3P)4
M
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(82 mg, 0.071 mmol). The vial was flushed with Ar(g), and then 1,4-
dioxane (3553 μL) and water (1.8 mL) were added in sequence. The
vial was sealed and heated to 80 °C overnight. The next morning, the
mixture was diluted with water (40 mL) and extracted with EtOAc (3
× 20 mL). The combined organic extracts were dried over sodium
sulfate, filtered, and evaporated. The residue was purified by
chromatography on silica gel (0−9% MeOH/DCM) to give (S)-4-
(2′-amino-7-hydroxy-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxa-
zole]-3-yl)benzonitrile (181.62 mg, 0.490 mmol, 69.0% yield) as a
over sodium sulfate, filtered, and evaporated. The residue was purified
by chromatography on silica gel (0−70% of a 90:10:1 mix of DCM/
MeOH/NH4OH in DCM) to afford (S)-2′-amino-3-morpholino-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazole]-7-yl trifluoromethanesul-
fonate (0.309 g, 0.635 mmol, 67% yield) as an off-white solid. The
material was about 80% pure as determined by LCMS and was used
directly in the next reaction. MS m/z = 487.2 [M + H]+. Calcd for
C19H18F3N4O6S: 487.1.
(S)-2′-Amino-3-(p-tolyl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-7-yl Trifluoromethanesulfonate (19d). A 25 mL flask was
charged with (S)-2′-amino-3-p-tolyl-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-7-ol (259.36 mg, 0.722 mmol) in DCM (7217
μL) to give an clear, orange solution. Triethylamine (201 μL, 1.443
mmol) and 1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)-
methanesulfonamide (271 mg, 0.758 mmol) were added in sequence.
The resulting mixture was stirred for 3 h and then was purified directly
by chromatography on silica gel (0−5% MeOH/DCM) to give (S)-2′-
amino-3-p-tolyl-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazole]-7-yl
trifluoromethanesulfonate (317.34 mg, 0.646 mmol, 89% yield) as a
cream-colored solid. 1H NMR (400 MHz, DMSO-d6) δ = 8.58 (d, J =
2.4 Hz, 1 H), 7.96 (d, J = 2.4 Hz, 1 H), 7.64−7.52 (m, 2 H), 7.49−
7.43 (m, 1 H), 7.37−7.30 (m, 2 H), 6.66 (br s, 2 H), 4.40−4.16 (m, 2
H), 2.36 (s, 3 H). MS m/z = 492.0 [M + H]+. Calcd for
C22H17F3N3O5S: 492.0.
(S)-2′-Amino-3-(4-cyanophenyl)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-7-yl Trifluoromethanesulfonate (19e). A
round-bottom flask was charged with (S)-4-(2′-amino-7-hydroxy-
5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazole]-3-yl)benzonitrile
(181.62 mg, 0.490 mmol) and DCM (4904 μL) to give a suspension.
Triethylamine (137 μL, 0.981 mmol) was added, and the starting
material dissolved after a few minutes of stirring. N-Phenyl-
bis(trifluoromethanesulfonimide) (184 mg, 0.515 mmol) was added,
and the mixture was stirred overnight. In the morning, the reaction
mixture was diluted with saturated aq sodium bicarbonate solution (20
mL). The layers were separated, and the aqueous layer was extracted
with DCM (2 × 20 mL). The combined organic extracts were dried
over sodium sulfate, filtered, and evaporated. The residue was purified
by chromatography on silica gel (0−5% MeOH/DCM) to give (S)-2′-
amino-3-(4-cyanophenyl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazole]-7-yl trifluoromethanesulfonate (186.48 mg, 0.371 mmol, 76%
yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ = 8.71
(d, J = 2.5 Hz, 1 H), 8.10 (d, J = 2.4 Hz, 1 H), 8.03−7.89 (m, 4 H),
7.59−7.53 (m, 1 H), 7.51−7.45 (m, 1 H), 7.34 (d, J = 2.9 Hz, 1 H),
6.65 (s, 2 H), 4.38 (d, J = 8.8 Hz, 1 H), 4.27 (d, J = 8.8 Hz, 1 H). MS
m/z = 502.8 [M + H]+. Calcd for C22H14F3N4O5S: 503.1.
1
yellow solid. H NMR (400 MHz, DMSO-d6) δ = 9.39 (s, 1 H), 8.65
(d, J = 2.5 Hz, 1 H), 8.05 (d, J = 2.4 Hz, 1 H), 8.00−7.86 (m, 4 H),
7.14−7.03 (m, 1 H), 6.81−6.70 (m, 2 H), 6.50 (br s, 2 H), 4.31−4.25
(m, 1 H), 4.23−4.18 (m, 1 H). MS m/z = 371.0 [M + H]+. Calcd for
C21H15N4O3: 371.1.
(S)-2′-Amino-3-(tetrahydro-2H-pyran-4-yl)-5′H-spiro[chromeno-
[2,3-b]pyridine-5,4′-oxazol]-7-yl Trifluoromethanesulfonate (19a).
A 25 mL round-bottom flask was charged with (S)-2′-amino-3-
(tetrahydro-2H-pyran-4-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-7-oland triethylamine (194 μL, 1.392 mmol) in DCM (2.5
mL) to give an opaque mixture. N-Phenyltrifluoromethanesulfonimide
(261 mg, 0.731 mmol) was added, and the resulting mixture was
stirred for 24 h. An additional portion of N-phenyltrifluoromethane-
sulfonimide (50 mg) was added, and the mixture was stirred for an
additional 4 h. The reaction mixture was diluted with DCM (20 mL)
and saturated aq sodium bicarbonate solution (20 mL). The layers
were separated, and the aqueous layer was extracted with DCM (2 ×
10 mL). The combined organic extracts were dried over sodium
sulfate, filtered, and evaporated. The residue was purified by
chromatography on silica gel (0−7% MeOH/DCM) to give (S)-2′-
amino-3-(tetrahydro-2H-pyran-4-yl)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazole]-7-yl trifluoromethanesulfonate (280.69 mg,
0.578 mmol, 83% yield) as a white solid. 1H NMR (400 MHz,
DMSO-d6) δ = 8.22 (d, J = 2.3 Hz, 1 H), 7.68 (br s, 1 H), 7.57−7.50
(m, 1 H), 7.46−7.41 (m, 1 H), 7.36 (br s, 1 H), 4.24 (br s, 2 H), 3.96
(dd, J = 1.7, 11.2 Hz, 2 H), 3.44 (tt, J = 2.5, 11.2 Hz, 2 H), 2.96−2.81
(m, 1 H), 1.75−1.59 (m, 4 H). MS m/z = 485.8 [M + H]+. Calcd for
C20H19F3N3O6S: 486.1.
(S)-2′-Amino-3-(6,6-dimethyl-3,6-dihydro-2H-pyran-4-yl)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-7-yl Trifluoromethane-
sulfonate (19b). (S)-2′-Amino-3-(6,6-dimethyl-3,6-dihydro-2H-pyran-
4-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-7-ol (0.231 g,
0.609 mmol) and triethylamine (0.255 mL, 1.827 mmol) were
combined in DCM (12.18 mL). 1,1,1-Trifluoro-N-phenyl-N-
(trifluoromethylsulfonyl)methanesulfonamide (0.239 g, 0.670 mmol)
was added, and the solution was stirred at room temperature
overnight. In the morning, the solution purified directly by
chromatography on silica gel (0−60% of a 90:10:1 mixture of
DCM/MeOH/NH4OH in DCM) to afford (S)-2′-amino-3-(6,6-
dimethyl-3,6-dihydro-2H-pyran-4-yl)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazole]-7-yl trifluoromethanesulfonate (0.258 g, 0.504
7-Bromo-3-iodo-5H-chromeno[2,3-b]pyridin-5-one (21). Step 1:
2-(4-Bromophenoxy)-5-iodonicotinic acid. A solution of 2,2,6,6-
tetramethylpiperidine (45.7 mL, 269 mmol) in THF (400 mL) was
cooled to −10 °C and then treated with n-butyllithium (103 mL, 257
mmol). After stirring for 10 min, the reaction mixture was cooled to
−78 °C. The cooled solution was treated with a solution of 2-fluoro-5-
iodopyridine (54.6 g, 245 mmol) in THF (200 mL). The reaction
mixture was stirred for 40 min, and then CO2 (g) gas was bubbled
through for 10 min via syringe. The reaction mixture was placed in a 0
°C bath, and CO2(g) was bubbled through for an additional 10 min. 4-
Bromophenol (46.6 g, 269 mmol) and potassium carbonate (42.3 g,
306 mmol) were added in sequence. The reaction mixture was
concentrated in vacuo, and the residue was suspended in DMF (500
mL). The resulting mixture was heated to 140 °C overnight. In the
morning, the mixture was cooled to room temperature and then
poured into 1N aq HCl. The resulting suspension was filtered, washed
with 1:1 water/acetone, and dried to give 2-(4-bromophenoxy)-5-
1
mmol, 83% yield) as a off-white solid. H NMR (400 MHz, DMSO-
d6) δ = 8.38 (d, J = 2.4 Hz, 1 H), 7.78 (s, 1 H), 7.54 (dd, J = 3.0, 9.0
Hz, 1 H), 7.48−7.40 (m, 1 H), 7.32 (br s, 1 H), 6.65 (br s, 2 H), 6.23
(s, 1 H), 4.33−4.12 (m, 2 H), 3.84 (t, J = 5.4 Hz, 2 H), 2.41−2.34 (m,
2 H), 1.28 (s, 6 H). MS m/z = 512.1 [M + H]+. Calcd for
C22H21F3N3O6S: 512.1.
(S)-2′-Amino-3-morpholino-5′H-spiro[chromeno[2,3-b]pyridine-
5,4′-oxazol]-7-yl Trifluoromethanesulfonate (19c). A 25 mL round-
bottom flask was charged with cesium carbonate (0.371 g, 1.138
mmol), (S)-2′-amino-3-morpholino-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-7-ol (0.336 g, 0.948 mmol), and DMF (4.74
mL). The resulting mixture was stirred for 10 min, and then the flask
was submerged in an ice bath for 5 min. N-Phenyltrifluoromethane-
sulfonimide (0.373 g, 1.043 mmol) was added as a single portion, and
the reaction was stirred overnight, during which the ice bath was
allowed to melt. In the morning, the mixture was cooled to 0 °C, and
additional portions of cesium carbonate (150 mg) and N-phenyl-
trifluoromethanesulfonimide were added in sequence. The mixture
was stirred for an additional 1 h and then was diluted with water and
extracted with EtOAc (2×). The combined organic extracts were dried
1
iodonicotinic acid (92.60 g, 220 mmol, 90% yield). H NMR (400
MHz, acetonitrile-d3) δ = 8.52 (d, J = 2.3 Hz, 1 H), 8.42 (d, J = 2.3 Hz,
1 H), 7.58−7.52 (m, 2 H), 7.08−7.04 (m, 2 H). MS m/z = 419.8 [M +
H]+. Calcd for C12H8BrINO: 419.9.
Step 2: 7-Bromo-3-iodo-5H-chromeno[2,3-b]pyridin-5-one. A flask
charged with 2-(4-bromophenoxy)-5-iodonicotinic acid (93 g, 220
mmol) and polyphosphoric acid 115% (926 g, 220 mmol) was heated
to 120 °C overnight. In the morning, starting material persisted, so the
N
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Journal of Medicinal Chemistry
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reaction mixture was heated to 140 °C for 3 h. The reaction mixture
was then cooled to room temperature and poured into water. The
water slurry was stirred for 2 h and then filtered. The collected solid
was washed with 1N aq NaOH, water, and 2-PrOH, then dried under
air to yield 7-bromo-3-iodo-5H-chromeno[2,3-b]pyridin-5-one (63.48
0 °C and was quenched with MeOH. The reaction mixture was poured
into saturated aq ammonium chloride solution and extracted with
EtOAc. The organic layer was washed with water and brine, dried over
MgSO4, and concentrated, yielding crude 7-bromo-3-(2-methoxy-2-
methylpropoxy)-5-methyl-5H-chromeno[2,3-b]pyridin-5-ol (1.80 g,
4.57 mmol, 56.1% yield). This material was used directly in the next
reaction. MS m/z = 394.0 [M + H]+. Calcd for C18H21BrNO4: 394.0.
rac-7-Bromo-3-(2-methoxy-2-methylpropoxy)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (24). A solution of
7-bromo-3-(2-methoxy-2-methylpropoxy)-5-methyl-5H-chromeno-
[2,3-b]pyridin-5-ol (1.780 g, 4.51 mmol) in THF (50 mL) was treated
with a 4 M HCl solution in 1,4-dioxane (0.113 mL, 0.451 mmol), and
the resulting mixture was heated to 50 °C for 1 h to afford a solution
containing the corresponding olefin. The solution was cooled to 0 °C.
Separately, silver cyanate (1.692 g, 11.29 mmol) was added in one
portion to a solution of iodine (1.260 g, 4.97 mmol) in THF (50 mL)
maintained at −40 °C. The resulting was stirred for 1 h at −40 °C, and
then the solution of olefin was via cannula. After an additional hour of
stirring, ammonia (13.54 mL of 2 M solution in 2-PrOH) was added in
one portion. The mixture was warmed to room temperature and
stirred for a further 3 h. The reaction mixture was quenched with a
10% aq sodium thiosulfate solution, and the resulting biphasic mixture
was stirred at room temperature for 1 h. The organic layer was
separated, washed with water, washed with brine, dried over MgSO4,
filtered, and concentrated. The crude product was purified by
chromatography on silica gel (0−10% MeOH/DCM) to give rac-
bromo-3-(2-methoxy-2-methylpropoxy)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-2′-amine (0.319 g, 0.735 mmol, 16.27% yield) as
an off-white solid. MS m/z = 434.0 [M + H]+. Calcd for
C19H21BrN3O4: 434.0.
3-Bromo-7-(2,2-dimethylpropoxy)-spiro[chromeno[2,3-b]-
pyridine]-5,4′-[1,3]oxazole]-2′-amine (rac-25). To a solution of 3-
bromo-7-hydroxy-spiro[chromeno[2,3-b]pyridine]-5,4′-[1,3]oxazole]-
2′-amine (650 mg, 1867 μmol) and DMF (7468 μL, 1867 μmol) in a
microwave vial were added cesium carbonate (1521 mg, 4668 μmol)
and 1-iodo-2,2-dimethylpropane (0.495 mL, 3.74 mmol). The mixture
was heated in a microwave at 100 °C for 1 h. An additional portion of
1-iodo-2,2-dimethylpropane (0.4 mL) was added, and the mixture was
heated in the microwave at 100 °C for another 1 h. The reaction was
diluted with water (5 mL) and EtOAc (5 mL) and stirred for 5 min
until homogeneous. The resulting mixture was poured into 10 mL of
ethyl acetate and 25 mL of saturated ammonium chloride, and the
layers were separated. The aqueous layer was extracted with ethyl
acetate (3 × 20 mL). The aqueous layer was then extracted with DCM
(3 × 15 mL). The organic layers were each washed with brine,
combined, dried over sodium sulfate, filtered, and concentrated. The
resulting oil was purified by silica gel chromatography (0−100%
EtOAc/hexanes) to provide 3-bromo-7-(2,2-dimethylpropoxy)-spiro-
[chromeno[2,3-b]pyridine]-5,4′-[1,3]oxazole]-2′-amine (375 mg, 48%
yield) as a yellow solid. MS m/z = 418.2 [M + H]+. Calcd for
C19H21BrN3O3: 418.1.
1
g, 158 mmol, 71.6% yield). H NMR (400 MHz, chloroform-d) δ =
8.98 (d, J = 2.4 Hz, 1 H), 8.94 (d, J = 2.4 Hz, 1 H), 8.44 (d, J = 2.4 Hz,
1 H), 7.89 (dd, J = 2.4, 8.9 Hz, 1 H), 7.53 (d, J = 9.0 Hz, 1 H). MS m/
z = 401.7 [M + H]+. Calcd for C12H6BrINO2: 401.7.
7-Bromo-3-hydroxy-5H-chromeno[2,3-b]pyridin-5-one (22). A
flask charged with Pd(OAc)2 (1.773 g, 7.90 mmol), X-Phos (11.29
g, 23.69 mmol), 7-bromo-3-iodo-5H-chromeno[2,3-b]pyridin-5-one
(63.5 g, 158 mmol), bis(pinacolato)diboron (42.1 g, 166 mmol), and
potassium phosphate (101 g, 474 mmol) was diluted with 1,4-dioxane
(600 mL), and the resulting mixture was stirred at room temperature
overnight. The reaction mixture was filtered through a plug of Celite,
and the filtrate was diluted with THF (500 mL) and a 6N aq sodium
hydroxide solution (132 mL, 790 mmol). The resulting mixture was
cooled to 0 °C, and hydrogen peroxide 30% (58.1 mL, 1895 mmol)
was added dropwise. The resulting mixture was stirred for 5 h and then
diluted with MeOH (500 mL). A 6N aq hydrochloric acid solution
(150 mL) and a 10% aq sodium thiosulfate solution (300 mL) were
added in sequence. The mixture was then concentrated in vacuo to
remove the volatiles, and the resulting suspension was filtered. The
collected solid was washed with 1:1 water/acetone and dried. The
resulting dark-brown solid was triturated with warm diethyl ether then
hot acetonitrile and then filtered to give 7-bromo-3-hydroxy-5H-
1
chromeno[2,3-b]pyridin-5-one (30.39 g, 104 mmol, 65.9% yield). H
NMR (400 MHz, chloroform-d) δ = 8.43 (d, J = 2.2 Hz, 1 H), 8.32 (d,
J = 2.8 Hz, 1 H), 8.03 (d, J = 2.7 Hz, 1 H), 7.83 (dd, J = 2.4, 8.8 Hz, 1
H), 7.54−7.47 (m, 1 H), 7.00 (s, 1 H). MS m/z = 291.9 [M + H]+.
Calcd for C12H7BrNO3: 291.9.
rac-7-Bromo-3-(2-methoxy-2-methylpropoxy)-5-methyl-5H-
chromeno[2,3-b]pyridin-5-ol (23). Step 1: A solution of 7-bromo-3-
hydroxy-5H-chromeno[2,3-b]pyridin-5-one (15.00 g, 51.4 mmol) in
DCM (100 mL) was treated with DBU (9.68 mL, 64.2 mmol) and
stirred for 10 min. 3-Bromo-2-methylpropene (5.44 mL, 53.9 mmol)
was added, and the resulting mixture was stirred for an additional hour.
The reaction mixture was quenched with 0.5 N aq citric acid solution
(200 mL) and concentrated in vacuo to remove the volatiles. The
resulting suspension was filtered, and the collected solid was washed
with 1:1 water/acetone and dried. The solid was purified by
chromatography on silica gel (0−50% EtOAc/hexane), yielding 7-
bromo-3-(2-methylallyloxy)-5H-chromeno[2,3-b]pyridin-5-one (4.50
1
g, 13.00 mmol, 25.3% yield). H NMR (400 MHz, acetonitrile-d3) δ
= 8.51 (d, J = 3.2 Hz, 1 H), 8.34 (d, J = 2.5 Hz, 1 H), 8.06 (d, J = 3.3
Hz, 1 H), 7.95 (dd, J = 2.5, 8.9 Hz, 1 H), 7.59 (d, J = 8.9 Hz, 1 H),
5.15 (d, J = 0.7 Hz, 1 H), 5.06 (d, J = 1.6 Hz, 1 H), 4.67 (s, 2 H), 1.85
(d, J = 0.5 Hz, 3 H). MS m/z = 346.0 [M + H]+. Calcd for
C16H13BrNO3: 346.0.
Step 2: A suspension of 7-bromo-3-((2-methylallyl)oxy)-5H-
chromeno[2,3-b]pyridin-5-one (2.420 g, 6.99 mmol) in MeOH (100
mL) was treated with N-iodosuccinimide (7.86 g, 35.0 mmol), and the
resulting mixture was stirred at room temperature for 36 h. The crude
product was purified by chromatography on silica gel (0−100%
EtOAc/hexane) to give 7-bromo-3-(3-iodo-2-methoxy-2-methylpro-
poxy)-5H-chromeno[2,3-b]pyridin-5-one (2.33 g, 4.62 mmol, 66.1%
(S)-3-(Neopentyloxy)-7-(pyrimidin-5-yl)-5′H-spiro[chromeno[2,3-
b]pyridine-5,4′-oxazol]-2′-amine (26). A sealable tube was charged
with (S)-7-bromo-3-(neopentyloxy)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-2′-amine (75 mg, 0.179 mmol), pyrimidin-5-
ylboronic acid (89 mg, 0.717 mmol), potassium carbonate (124 mg,
0.897 mmol), PdCl2(AmPhos)2 (6.35 mg, 8.97 μmol), THF (1793
μL), and water (1 mL). The tube was flushed with Ar(g), sealed, and
heated to 110 °C for 1 h. The reaction was diluted with water (25 mL)
and poured into a separatory funnel containing ethyl acetate (25 mL).
The layers were separated, and the aqueous layer was extracted with
ethyl acetate (2 × 10 mL). The combined organic layers were washed
with brine, dried over sodium sulfate, filtered, and concentrated in
vacuo to provide a brown foam. This residue was purified by silica gel
chromatography (0−10% MeOH/DCM with 0.1% ammonium
hydroxide) to provide a tan solid. This solid was purified further by
chromatography on silica gel (0−100% EtOAc/DCM) to provide (S)-
3-(neopentyloxy)-7-(pyrimidin-5-yl)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-2′-amine (54 mg, 0.129 mmol, 72.1% yield) as a
light-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ = 9.19 (s, 1 H),
1
yield) as an oil. H NMR (400 MHz, acetonitrile-d3) δ = 8.52 (d, J =
3.3 Hz, 1 H), 8.34 (d, J = 2.4 Hz, 1 H), 8.13−8.10 (m, 1 H), 7.95 (dd,
J = 2.5, 8.9 Hz, 1 H), 7.60 (d, J = 9.0 Hz, 1 H), 4.24−4.07 (m, 2 H),
3.62−3.52 (m, 2 H), 3.27 (s, 3 H), 1.45 (s, 3 H). MS m/z = 503.9 [M
+ H]+. Calcd for C17H16BrINO4: 503.9.
Step 3: A solution of 7-bromo-3-(3-iodo-2-methoxy-2-methylpro-
poxy)-5H-chromeno[2,3-b]pyridin-5-one (4.10 g, 8.13 mmol) in THF
(100 mL) cooled to −40 °C was treated with methylmagnesium
chloride (5.42 mL of 3 M solution in diethyl ether, 16.27 mmol). After
stirring for 2 h, the reaction mixture was allowed to warm to room
temperature. Lithium triethylborohydride (40.7 mL of a 1 M solution
in THF, 40.7 mmol) was added added over 5 min. After stirring for an
additional 2 h at room temperature, the reaction mixture was cooled to
O
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9.10 (s, 2 H), 8.01 (d, J = 2.9 Hz, 1 H), 7.79 (dd, J = 2.3, 8.5 Hz, 1 H),
7.67 (d, J = 2.2 Hz, 1 H), 7.36 (d, J = 8.5 Hz, 1 H), 7.30 (d, J = 3.0 Hz,
1 H), 6.51 (br s, 2 H), 4.32 (s, 2 H), 3.79−3.65 (m, 2 H), 1.02 (s, 9
H). MS m/z = 418.2 [M + H]+. Calcd for C23H24N5O3: 418.2.
(S)-3-(Neopentyloxy)-7-(pyridin-3-yl)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-2′-amine (27). A sealable tube was charged with
(S)-7-bromo-3-(neopentyloxy)-5′H-spiro[chromeno[2,3-b]pyridine-
5,4′-oxazol]-2′-amine (150 mg, 0.359 mmol), 3-pyridylboronic acid
(132 mg, 1.076 mmol), potassium carbonate (248 mg, 1.793 mmol),
tetrakis(triphenylphosphine)palladium(0) (20.72 mg, 0.018 mmol),
THF (3586 μL), and water (0.5 mL). The tube was flushed with
Ar(g), sealed, and heated to 110 °C for 1 h. The reaction was diluted
with water (25 mL) and poured into a separatory funnel containing
ethyl acetate (50 mL). The layers were separated, and the aqueous
layer was extracted with ethyl acetate (2 × 10 mL). The combined
organic layers were washed with brine, dried over sodium sulfate,
filtered, and concentrated in vacuo to provide a brown foam. This
residue was purified by silica gel chromatography (0−10% MeOH/
DCM with 0.1% ammonium hydroxide) to provide (S)-3-(neo-
pentyloxy)-7-(pyridin-3-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-2′-amine (116 mg, 0.279 mmol, 78% yield) as a light-yellow
foam. 1H NMR (400 MHz, DMSO-d6) δ = 8.85 (d, J = 1.9 Hz, 1 H),
8.57 (dd, J = 1.6, 4.8 Hz, 1 H), 8.06−7.95 (m, 2 H), 7.71 (dd, J = 2.3,
8.5 Hz, 1 H), 7.58 (d, J = 2.2 Hz, 1 H), 7.50 (dd, J = 4.8, 7.9 Hz, 1 H),
7.35−7.29 (m, 2 H), 6.52 (s, 2 H), 4.35−4.22 (m, 2 H), 3.77−3.65
(m, 2 H), 1.02 (s, 9 H). MS m/z = 417.2 [M + H]+. Calcd for
C24H25N4O3: 417.2.
(S)-7-(2-Fluoropyridin-3-yl)-3-(neopentyloxy)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (28). A sealable
tube was charged with (S)-7-bromo-3-(neopentyloxy)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (104822−28) (75
mg, 0.179 mmol), 2-fluoropyridin-3-ylboronic acid (76 mg, 0.538
mmol), potassium carbonate (124 mg, 0.897 mmol), PdCl2(AmPhos)2
(6.35 mg, 8.97 μmol), THF (1793 μL), and water (1 mL). The tube
was flushed with Ar(g), sealed, and heated to 110 °C for 1 h. The
reaction was diluted with water (25 mL) and poured into a separatory
funnel containing ethyl acetate (25 mL). The layers were separated,
and the aqueous layer was extracted with ethyl acetate (2 × 10 mL).
The combined organic layers were washed with brine, dried over
sodium sulfate, filtered, and concentrated in vacuo to provide a brown
foam. This residue was purified by silica gel chromatography (0−10%
MeOH/DCM with 0.1% ammonium hydroxide) to provide (S)-7-(2-
fluoropyridin-3-yl)-3-(neopentyloxy)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-2′-amine (70 mg, 0.161 mmol, 90% yield) as a
light-yellow solid. 1H NMR (400 MHz, DMSO-d6) δ = 8.24 (d, J = 4.7
Hz, 1 H), 8.15−8.07 (m, 1 H), 8.00 (d, J = 3.0 Hz, 1 H), 7.61 (d, J =
8.9 Hz, 1 H), 7.54 (s, 1 H), 7.49 (ddd, J = 1.9, 5.0, 7.2 Hz, 1 H), 7.35−
7.27 (m, 2 H), 6.53 (s, 2 H), 4.30−4.20 (m, 2 H), 3.78−3.66 (m, 2 H),
1.02 (s, 9 H). MS m/z = 435.0 [M + H]+. Calcd for C24H24FN4O3:
435.1.
(S)-7-(2,2-Dimethylpropoxy)-3-(5-pyrimidinyl)-spiro[chromeno-
[2,3-b]pyridine]-5,4′-[1,3]oxazole]-2′-amine (29). A sealable tube was
charged with 3-bromo-7-(2,2-dimethylpropoxy)-spiro[chromeno[2,3-
b]pyridine]-5,4′-[1,3]oxazole]-2′-amine (0.120 g, 287 μmol), pyrimi-
din-5-ylboronic acid (98 mg, 789 μmol), Pd(Ph3P)4 (33 mg, 29 μmol),
THF (8 mL), and an aqueous solution of potassium carbonate (1 M)
(1434 μL, 1434 μmol). The tube was sealed and heated at 90 °C for
2.5 h. The reaction was cooled to room temperature and diluted with
water (15 mL). The organics were removed, and the aqueous layer was
extracted with ethyl acetate (3 × 45 mL). The combined organics were
washed with brine, dried over sodium sulfate, filtered, and
concentrated to provide a residue which was purified by chromatog-
raphy on silica gel (0−10% MeOH/DCM) to provide rac-7-(2,2-
dimethylpropoxy)-3-(5-pyrimidinyl)-spiro[chromeno[2,3-b]pyridine]-
5,4′-[1,3]oxazole]-2′-amine as a yellow solid. MS m/z = 418.2 [M +
H]+. Calcd for C23H24N5O3: 418.2. Chiral separation of racemic 7-
(2,2-dimethylpropoxy)-3-(5-pyrimidinyl)-spiro[chromeno[2,3-b]-
pyridine]-5,4′-[1,3]oxazole]-2′-amine: Racemic 7-(2,2-dimethylpro-
poxy)-3-(5-pyrimidinyl)-spiro[chromeno[2,3-b]pyridine]-5,4′-[1,3]-
oxazole]-2′-amine (69 mg) was subjected to SFC using 15:85:0.1
MeOH:CO2:DEA at 70 mL/min on a 2 cm × 15 cm, 5 μm ChiralPak
AD-H column and 100 bar system pressure. The first peak (RT = 3.2
min) provided (S)-7-(2,2-dimethylpropoxy)-3-(5-pyrimidinyl)-spiro-
[chromeno[2,3-b]pyridine]-5,4′-[1,3]oxazole]-2′-amine (29 mg, 24%
yield, >99% ee), and the second peak (RT = 6.8 min) provided (R)-7-
(2,2-dimethylpropoxy)-3-(5-pyrimidinyl)-spiro[chromeno[2,3-b]-
pyridine]-5,4′-[1,3]oxazole]-2′-amine (>99% ee). 1H NMR (400
MHz, DMSO-d6) δ = 9.22 (s, 1 H), 9.18 (s, 2 H), 8.69 (d, J = 2.4
Hz, 1 H), 8.15 (d, J = 2.4 Hz, 1 H), 7.19 (d, J = 8.9 Hz, 1 H), 6.98 (dd,
J = 3.0, 8.9 Hz, 1 H), 6.81 (d, J = 2.9 Hz, 1 H), 6.52 (s, 2 H), 4.35−
4.22 (m, 2 H), 3.62 (q, J = 8.6 Hz, 2 H), 1.01 (s, 9 H).
(S)-7-(2-Fluoropyridin-3-yl)-3-(2-methoxy-2-methylpropoxy)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (30). A vial
was charged with 2-fluoropyridin-3-ylboronic acid (0.156 g, 1.105
mmol), tetrakis(triphenylphosphine)palladium(0) (0.043 g, 0.037
mmol), potassium carbonate (0.255 g, 1.842 mmol), rac-7-bromo-3-
(2-methoxy-2-methylpropoxy)-5′H-spiro[chromeno[2,3-b]pyridine-
5,4′-oxazol]-2′-amine (0.160 g, 0.368 mmol), THF (3 mL), and water
(0.5 mL). The resulting mixture was heated to 110 °C for 2 h. The
reaction mixture was cooled to room temperature and extracted with
EtOAc. The organic layer was dried over MgSO4, filtered, and
concentrated. The residue was purified by chromatography on silica
gel (0−10% MeOH/DCM) to give rac-7-(2-fluoropyridin-3-yl)-3-(2-
methoxy-2-methylpropoxy)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-2′-amine (0.072 g, 0.160 mmol, 43.4% yield). This material
was dissolved in methanol, and the resulting solution was purified by
SFC, using 1.5 mL injections on a 5 μm, 3 cm × 15 cm Chiralpak IC
column with 40% MeOH/0.2% diethylamine/60% CO2 at a flow rate
of 100 mL/min. This afforded peak 1, (S)-7-(2-fluoropyridin-3-yl)-3-
(2-methoxy-2-methylpropoxy)-5′H-spiro[chromeno[2,3-b]pyridine-
5,4′-oxazol]-2′-amine (0.026 g, 0.058 mmol, 15.67% yield), and peak
2, (R)-7-(2-fluoropyridin-3-yl)-3-(2-methoxy-2-methylpropoxy)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (0.026 g, 0.058
1
mmol, 15.67% yield). Data for the title compound. H NMR (400
MHz, acetonitrile-d3) δ = 8.18 (td, J = 1.6, 4.8 Hz, 1 H), 8.00 (ddd, J =
1.9, 7.5, 10.2 Hz, 1 H), 7.96 (d, J = 2.9 Hz, 1 H), 7.60−7.58 (m, 1 H),
7.57−7.55 (m, 1 H), 7.41−7.35 (m, 2 H), 7.28 (dd, J = 0.7, 8.2 Hz, 1
H), 5.08 (br s, 2 H), 4.32 (d, J = 1.8 Hz, 2 H), 3.93 (d, J = 1.0 Hz, 2
H), 3.23 (s, 3 H), 1.27 (s, 6 H). MS m/z = 451.2 [M + H]+. Calcd for
C24H24FN4O4: 451.2.
(S)-3-(3,6-Dihydro-2H-pyran-4-yl)-7-(2-fluoropyridin-3-yl)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (31). A vial
was charged with (S)-3-bromo-7-(2-fluoropyridin-3-yl)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (0.150 g, 0.351
mmol), Pd(Ph3P)4 (0.020 g, 0.018 mmol), potassium carbonate
(0.243 g, 1.756 mmol), 1,4-dioxane (1.756 mL), and water (0.585
mL). The vial was flushed with argon, sealed, and heated overnight at
80 °C. The mixture was cooled to room temperature and then diluted
water and extracted with ethyl acetate (2×). The combined organic
extracts were dried with sodium sulfate, filtered, and concentrated. The
residue was purified by chromatography on silica gel (0−6% MeOH/
DCM0 to afford (S)-3-(3,6-dihydro-2H-pyran-4-yl)-7-(2-fluoropyri-
din-3-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine
1
(0.089 g, 0.207 mmol, 58.9% yield) as a white solid. H NMR (400
MHz, DMSO-d6) δ = 8.39 (d, J = 2.4 Hz, 1 H), 8.25 (td, J = 1.5, 4.9
Hz, 1 H), 8.12 (ddd, J = 1.9, 7.5, 10.3 Hz, 1 H), 7.79 (d, J = 2.4 Hz, 1
H), 7.62 (td, J = 1.9, 8.5 Hz, 1 H), 7.56 (t, J = 1.8 Hz, 1 H), 7.49 (ddd,
J = 1.9, 5.0, 7.3 Hz, 1 H), 7.37 (d, J = 8.5 Hz, 1 H), 6.51 (s, 2 H), 6.31
(t, J = 1.5 Hz, 1 H), 4.28 (s, 2 H), 4.25 (q, J = 2.6 Hz, 2 H), 3.85 (t, J =
5.5 Hz, 2 H). MS m/z = 431.1 [M + H]+. Calcd for C24H20FN4O3:
431.1.
(S)-3-(5,6-Dihydro-2H-pyran-3-yl)-7-(2-fluoropyridin-3-yl)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (32). A vial
was charged with (S)-3-bromo-7-(2-fluoropyridin-3-yl)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (129 mg, 0.302
mmol), 2-(5,6-dihydro-2H-pyran-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxa-
borolane (307 mg, 0.453 mmol), potassium carbonate (209 mg, 1.510
mmol), and PdCl2(dppf)−CH2Cl2 (12.33 mg, 0.015 mmol). The vial
was flushed with Ar(g), and then 1,4-dioxane (1510 μL) and water
(0.5 mL) were added in sequence. The vial was sealed and heated to
P
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70 °C for 40 min. The mixture was diltued with EtOAc (15 mL) and
water (15 mL). The layers were separated, and the aqueous layer was
extracted with EtOAc (15 mL). The combined organic extracts were
dried over sodium sulfate, filtered, and evaporated. The residue was
purified by chromatography on silica gel (0−50% of a 90:10:1 mix of
DCM/MeOH/NH4OH in DCM) to give ca. 60 mg of a brown solid.
The solid was dissolved in MeOH, and the resulting solution was
loaded onto a 2-g SCX acidic ion exchange column. The column was
eluted with MeOH, then with 2N ammonia in methanol. The basic
fraction was concentrated to give (S)-3-(5,6-dihydro-2H-pyran-3-yl)-7-
(2-fluoropyridin-3-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxa-
zol]-2′-amine (52.48 mg, 0.122 mmol, 40.4% yield) as an off-white
solid. 1H NMR (400 MHz, DMSO-d6) δ = 8.30 (d, J = 2.4 Hz, 1 H),
8.25 (td, J = 1.5, 4.8 Hz, 1 H), 8.11 (ddd, J = 2.0, 7.5, 10.3 Hz, 1 H),
7.73 (d, J = 2.4 Hz, 1 H), 7.62 (td, J = 1.7, 8.8 Hz, 1 H), 7.56 (t, J = 1.8
Hz, 1 H), 7.49 (ddd, J = 1.9, 5.0, 7.2 Hz, 1 H), 7.36 (d, J = 8.5 Hz, 1
H), 6.53 (br s, 2 H), 6.32 (td, J = 2.2, 3.9 Hz, 1 H), 4.45 (qd, J = 2.3,
6.6 Hz, 2 H), 4.28 (s, 2 H), 3.76 (t, J = 5.5 Hz, 2 H), 2.28 (qd, J = 2.7,
6.8 Hz, 2 H). MS m/z = 431.0 [M + H]+. Calcd for C24H20FN4O3:
431.1.
(S)-7-(2-Fluoropyridin-3-yl)-3-(tetrahydro-2H-pyran-4-yl)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (33). A vial
was charged with (S)-2′-amino-3-(tetrahydro-2H-pyran-4-yl)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazole]-7-yl trifluoromethanesul-
fonate (70.0 mg, 0.144 mmol), 2-fluoropyridin-3-ylboronic acid (61.0
mg, 0.433 mmol), potassium carbonate (100 mg, 0.721 mmol), and
Pd(Ph3P)4 (8.33 mg, 7.21 μmol). The vial was flushed with Ar(g), and
then 1,4-dioxane (721 μL) and water (0.3 mL) were added in
sequence. The vial was sealed and heated to 80 °C for 50 min. The
mixture was diluted with brine (20 mL) and extracted with EtOAc (2
× 15 mL). The combined organic extracts were dried over sodium
sulfate, filtered, and evaporated. The residue was purified by
chromatography on silica gel (0−70% of a 90:10:1 mix of DCM/
MeOH/NH4OH in DCM) to give (S)-7-(2-fluoropyridin-3-yl)-3-
(tetrahydro-2H-pyran-4-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-
oxazol]-2′-amine (60.4 mg, 0.140 mmol, 97% yield) as a white solid.
1H NMR (400 MHz, DMSO-d6) δ = 8.24 (d, J = 4.8 Hz, 1 H), 8.20 (d,
(m, 2 H), 1.28 (s, 6 H). MS m/z = 459.3 [M + H]+. Calcd for
C26H24FN4O3: 459.2.
(S)-7-(2-Fluoropyridin-3-yl)-3-morpholino-5′H-spiro[chromeno-
[2,3-b]pyridine-5,4′-oxazol]-2′-amine (35). A vial was charged (S)-2′-
amino-3-morpholino-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxa-
zole]-7-yl trifluoromethanesulfonate (0.060 g, 0.123 mmol), 2-
fluoropyridin-3-ylboronic acid (0.052 g, 0.370 mmol), potassium
carbonate (0.085 g, 0.617 mmol), and Pd(Ph3P)4 (7.13 mg, 6.17
μmol). The vial was flushed with Ar(g) and then 1,4-dioxane (0.617
mL) and water (0.36 mL). The vial was sealed and heated at 75 °C for
1 h. The reaction mixture was diluted with ethyl acetate and washed
with water. The aqueous layer was extracted with ethyl acetate, and the
combined organic extracts were washed with brine, dried over sodium
sulfate, filtered, and concentrated. The material was purified via
chromatography on silica gel (0−10% MeOH/DCM) to give partially
purified material. This material was purified further by reverse-phase
HPLC (20−90% CH3CN/H2O with 0.1% TFA). Fractions containing
clean desired product were combined, and the combined solution was
partitioned between DCM and saturated aq sodium bicarbonate
solution. The aqueous layer was extracted with DCM, and the
combined organic extracts were dried with sodium sulfate, filtered, and
concentrated to afford (S)-7-(2-fluoropyridin-3-yl)-3-morpholino-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (0.018 g, 0.042
mmol, 33.7% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6)
δ = 8.24 (td, J = 1.6, 4.8 Hz, 1 H), 8.10 (ddd, J = 1.9, 7.5, 10.4 Hz, 1
H), 8.00 (d, J = 2.9 Hz, 1 H), 7.60 (td, J = 1.7, 8.8 Hz, 1 H), 7.53 (t, J
= 1.8 Hz, 1 H), 7.49 (ddd, J = 1.9, 5.0, 7.3 Hz, 1 H), 7.35−7.28 (m, 2
H), 6.50 (s, 2 H), 4.24 (s, 2 H), 3.77 (t, J = 4.8 Hz, 4 H), 3.16−3.08
(m, 4 H). MS m/z = 434.4 [M + H]+. Calcd for C23H21FN5O3: 434.2.
(S)-7-(2-Fluoropyridin-3-yl)-3-(p-tolyl)-5′H-spiro[chromeno[2,3-
b]pyridine-5,4′-oxazol]-2′-amine (36). A vial was charged with (S)-
2′-amino-3-p-tolyl-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazole]-
7-yl trifluoromethanesulfonate (45.0 mg, 0.092 mmol), 2-fluoropyr-
idin-3-ylboronic acid (38.7 mg, 0.275 mmol), potassium carbonate
(229 μL, 0.458 mmol), and Pd(Ph3P)4 (5.29 mg, 4.58 μmol). The vial
was flushed with Ar(g), and then 1,4-dioxane (1 mL) and water (0.5
mL) were added in sequence. The vial was sealed and heated to 80 °C
for 1 h. The mixture was diluted with EtOAc, washed with brine, dried
over sodium sulfate, filtered, and evaporated. The residue was purified
by chromatography on silica gel (0−5% MeOH/DCM) to give (S)-7-
(2-fluoropyridin-3-yl)-3-p-tolyl-5′H-spiro[chromeno[2,3-b]pyridine-
5,4′-oxazol]-2′-amine (29.53 mg, 0.067 mmol, 73.6% yield) as a tan
J = 2.2 Hz, 1 H), 8.11 (ddd, J = 1.9, 7.7, 10.1 Hz, 1 H), 7.66 (d, J = 2.2
Hz, 1 H), 7.61 (d, J = 8.6 Hz, 1 H), 7.56 (s, 1 H), 7.49 (ddd, J = 1.8,
5.0, 7.2 Hz, 1 H), 7.35 (d, J = 8.5 Hz, 1 H), 6.51 (s, 2 H), 4.28−4.21
(m, 2 H), 4.00−3.93 (m, 2 H), 3.50−3.39 (m, 2 H), 2.95−2.84 (m, 1
H), 1.79−1.60 (m, 4 H). MS m/z = 433.0 [M + H]+. Calcd for
C24H22FN4O3: 433.2.
1
solid. H NMR (400 MHz, DMSO-d6) δ = 8.57 (d, J = 2.4 Hz, 1 H),
8.25 (td, J = 1.6, 4.8 Hz, 1 H), 8.13 (ddd, J = 1.9, 7.5, 10.3 Hz, 1 H),
7.97 (d, J = 2.4 Hz, 1 H), 7.67−7.56 (m, 4 H), 7.50 (ddd, J = 1.9, 5.0,
7.3 Hz, 1 H), 7.39 (d, J = 8.5 Hz, 1 H), 7.33 (d, J = 7.8 Hz, 2 H), 6.54
(s, 2 H), 4.41−4.25 (m, 2 H), 2.36 (s, 3 H). MS m/z = 439.0 [M +
H]+. Calcd for C26H20FN4O2: 439.2.
(S)-3-(6,6-Dimethyl-3,6-dihydro-2H-pyran-4-yl)-7-(2-fluoropyri-
din-3-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine
(34). A vial was charged with (S)-2′-amino-3-(6,6-dimethyl-3,6-
dihydro-2H-pyran-4-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxa-
zole]-7-yl trifluoromethanesulfonate (0.130 g, 0.254 mmol), 2-
fluoropyridin-3-ylboronic acid (0.107 g, 0.763 mmol), and Pd(Ph3P)4
(0.015 g, 0.013 mmol). The vial was purged with Ar(g), and then
DMF (1.271 mL) and potassium carbonate (0.635 mL, 1.271 mmol)
(as a 2.0 M aq solution) were added in sequence. The vial was sealed
and stirred at 70 °C overnight. In the morning, the reaction was
diluted with ethyl acetate and washed with water. The aqueous layer
was extracted with ethyl acetate, and the combined organic layers were
dried with sodium sulfate, filtered, and concentrated. The material was
purified via chromatography on silica gel (0−5% MeOH/DCM) afford
30 mg of product as an off-white solid. The impure fractions were
combined and repurified via chromatography on silica gel (0−5%
MeOH/DCM). Fractions containing clean desired product were
combined with the previously isolated material and concentrated to
afford (S)-3-(6,6-dimethyl-3,6-dihydro-2H-pyran-4-yl)-7-(2-fluoropyr-
idin-3-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine
(SS)-4-(2′-Amino-7-(2-fluoropyridin-3-yl)-5′H-spiro[chromeno-
[2,3-b]pyridine-5,4′-oxazol]-3-yl)benzonitrile (37). A vial was charged
with (S)-2′-amino-3-(4-cyanophenyl)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazole]-7-yl trifluoromethanesulfonate (50 mg, 0.100
mmol), 2-fluoropyridin-3-ylboronic acid (42.1 mg, 0.299 mmol),
potassium carbonate (68.8 mg, 0.498 mmol), and Pd(Ph3P)4 (5.75 mg,
4.98 μmol). The vial was flushed with Ar(g), and then 1,4-dioxane
(498 μL) and water (0.25 mL) were added in sequence. The vial was
sealed and heated to 80 °C for 2.5 h. The mixture was diluted with
EtOAc (20 mL), washed with water (15 mL), dried over sodium
sulfate, filtered, and evaporated. The residue was purified by
chromatography on silica gel (0−100% of a 90:10:1 mix of DCM/
MeOH/NH4OH in DCM) to give (S)-4-(2′-amino-7-(2-fluoropyr-
idin-3-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazole]-3-yl)-
1
benzonitrile (35.91 mg, 0.080 mmol, 80% yield) as a white solid. H
NMR (400 MHz, DMSO-d6) δ = 8.70 (d, J = 2.4 Hz, 1 H), 8.28−8.23
(m, 1 H), 8.17−8.07 (m, 2 H), 8.02−7.89 (m, 4 H), 7.67−7.62 (m, 1
H), 7.58 (t, J = 1.8 Hz, 1 H), 7.50 (ddd, J = 2.0, 4.9, 7.4 Hz, 1 H), 7.41
(d, J = 8.5 Hz, 1 H), 6.54 (s, 2 H), 4.45−4.26 (m, 2 H). MS m/z =
450.0 [M + H]+. Calcd for C26H17FN5O2: 450.1.
1
(0.044 g, 0.096 mmol, 37.8% yield) as a white solid. H NMR (400
MHz, DMSO-d6) δ = 8.37 (d, J = 2.4 Hz, 1 H), 8.25 (td, J = 1.5, 4.8
Hz, 1 H), 8.11 (ddd, J = 1.9, 7.5, 10.3 Hz, 1 H), 7.78 (d, J = 2.5 Hz, 1
H), 7.62 (td, J = 1.7, 8.8 Hz, 1 H), 7.56 (t, J = 1.8 Hz, 1 H), 7.49 (ddd,
J = 2.0, 5.0, 7.3 Hz, 1 H), 7.36 (d, J = 8.5 Hz, 1 H), 6.52 (s, 2 H), 6.22
(t, J = 1.4 Hz, 1 H), 4.28 (s, 2 H), 3.84 (t, J = 5.4 Hz, 2 H), 2.41−2.35
(S)-7-(2-Fluoropyridin-3-yl)-3-(2-fluoropyridin-4-yl)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (38). A vial was
Q
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charged with (S)-3-bromo-7-(2-fluoropyridin-3-yl)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (70 mg, 0.164
mmol), 2-fluoropyridin-4-ylboronic acid (69.3 mg, 0.492 mmol),
potassium carbonate (113 mg, 0.819 mmol), and Pd(Ph3P)4 (9.47 mg,
8.19 μmol). The vial was flushed with Ar(g), and then 1,4-dioxane
(819 μL) and water (1 mL) were added in sequence. The vial was
sealed and heated to 80 °C for 3 h. The mixture was cooled and
diluted with EtOAc (15 mL) and brine (15 mL). The layers were
separated, and the aqueous layer was extracted with EtOAc (15 mL).
The combined organic extracts were dried over sodium sulfate, filtered,
and evaporated. The residue was purified by chromatography on silica
gel (0−70% of a 90:10:1 mix of DCM/MeOH/NH4OH in DCM) to
afford (S)-7-(2-fluoropyridin-3-yl)-3-(2-fluoropyridin-4-yl)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (31.26 mg, 0.070
Hz, 1 H), 7.57 (d, J = 1.7 Hz, 1 H), 7.49 (ddd, J = 1.9, 5.1, 7.3 Hz, 1
H), 7.38 (d, J = 8.5 Hz, 1 H), 6.58 (br s, 2 H), 4.34−4.29 (m, 1 H),
4.28−4.22 (m, 1 H), 3.33 (s, 3 H), 1.50 (s, 6 H). MS m/z = 445.0 [M
+ H]+. Calcd for C25H22FN4O3: 445.2.
(S)-4-(2′-Amino-7-(2-fluoropyridin-3-yl)-5′H-spiro[chromeno[2,3-
b]pyridine-5,4′-oxazol]-3-yl)-2-methylbut-3-yn-2-ol (41). A vial was
charged with (S)-3-bromo-7-(2-fluoropyridin-3-yl)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (101 mg, 0.237
mmol), Pd(Ph3P)4 (27.4 mg, 0.024 mmol), CuI (4.51 mg, 0.024
mmol), THF (0.95 mL), and DMF (0.95 mL). N,N-Diisopropylamine
(664 μL, 4.74 mmol) and 2-methylbut-3-yn-2-ol (116 μL, 1.184
mmol) were added in sequence. The vial was flushed with Ar(g),
sealed, and heated at 110 °C for 2.5 h. The reaction mixture was
diluted with water and extracted with EtOAc (3×). The combined
organic extracts were dried over sodium sulfate, filtered, and
evaporated. The residue was purified by chromatography on silica
gel (0−100% of a 90:10:1 mixture of DCM/MeOH/NH4OH) to give
(S)-4-(2′-amino-7-(2-fluoropyridin-3-yl)-5′H-spiro[chromeno[2,3-b]-
pyridine-5,4′-oxazol]-3-yl)-2-methylbut-3-yn-2-ol (36.4 mg, 60%
1
mmol, 43.0% yield) as a white solid. H NMR (400 MHz, DMSO-
d6) δ = 8.81 (d, J = 2.4 Hz, 1 H), 8.36 (d, J = 5.4 Hz, 1 H), 8.26 (td, J
= 1.5, 4.8 Hz, 1 H), 8.23 (d, J = 2.4 Hz, 1 H), 8.16−8.10 (m, 1 H),
7.76 (td, J = 1.8, 5.3 Hz, 1 H), 7.68−7.63 (m, 2 H), 7.57 (t, J = 1.8 Hz,
1 H), 7.50 (ddd, J = 1.9, 5.0, 7.3 Hz, 1 H), 7.42 (d, J = 8.5 Hz, 1 H),
6.52 (s, 2 H), 4.45−4.41 (m, 1 H), 4.39−4.36 (m, 1 H). MS m/z =
444.0 [M + H]+. Calcd for C24H16F2N5O2: 444.1.
1
yield) as an off-white solid. H NMR (400 MHz, DMSO-d6) δ =
8.31 (d, J = 2.2 Hz, 1 H), 8.25 (td, J = 1.5, 4.8 Hz, 1 H), 8.12 (ddd, J =
2.0, 7.5, 10.4 Hz, 1 H), 7.71 (d, J = 2.2 Hz, 1 H), 7.63 (td, J = 1.7, 8.8
Hz, 1 H), 7.58 (t, J = 1.8 Hz, 1 H), 7.49 (ddd, J = 2.0, 5.0, 7.3 Hz, 1
H), 7.37 (d, J = 8.5 Hz, 1 H), 6.58 (s, 2 H), 5.54 (s, 1 H), 4.32−4.28
(m, 1 H), 4.23 (d, J = 8.7 Hz, 1 H), 1.49 (s, 6 H). MS m/z = 431.0 [M
+ H]+. Calcd for C24H20FN4O3: 431.2.
(S)-3-(3,3-Dimethylbut-1-yn-1-yl)-7-(2-fluoropyridin-3-yl)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (39). A vial
was charged with (S)-3-bromo-7-(2-fluoropyridin-3-yl)-5′H-spiro-
[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (80 mg, 0.187
mmol), Pd(Ph3P)4 (21.64 mg, 0.019 mmol), CuI (3.57 mg, 0.019
mmol), THF (0.75 mL), and DMF (0.75 mL). N,N-Diisopropylamine
(525 μL, 3.75 mmol) and 3,3-dimethylbut-1-yne (115 μL, 0.936
mmol) were added in sequence. The vial was flushed with Ar(g),
sealed, and heated to 110 °C for 3 h. The mixture was partitioned
between water (10 mL) and EtOAc (10 mL). The layers were
separated, and the aqueous layer was extracted with EtOAc (2 × 10
mL). The combined organic extracts were dried over sodium sulfate,
filtered, and evaporated. The residue was purified by chromatography
on silica gel (0−70% of a 90:10:1 mixture of DCM/MeOH/NH4OH
in DCM) to give 80 mg of a solid. This solid was dissolved in MeOH
and purified further by reverse-phase HPLC (10−90% CH3CN/H2O
with 0.1% TFA). The fractions containing product were combined in
saturated aq sodium bicarbonate solution with the aid of methanol and
extracted with DCM (3×). The combined organic extracts were dried
over sodium sulfate, filtered, and evaporated to give (S)-3-(3,3-
dimethylbut-1-ynyl)-7-(2-fluoropyridin-3-yl)-5′H-spiro[chromeno-
[2,3-b]pyridine-5,4′-oxazol]-2′-amine (46.61 mg, 0.109 mmol, 58.1%
yield) as a white powder. 1H NMR (500 MHz, DMSO-d6) δ = 8.27 (d,
J = 2.2 Hz, 1 H), 8.25 (td, J = 1.4, 4.8 Hz, 1 H), 8.12 (ddd, J = 1.9, 7.5,
10.3 Hz, 1 H), 7.67 (d, J = 2.3 Hz, 1 H), 7.65−7.61 (m, 1 H), 7.58−
7.56 (m, 1 H), 7.49 (ddd, J = 1.8, 5.0, 7.2 Hz, 1 H), 7.36 (d, J = 8.5 Hz,
1 H), 6.55 (s, 2 H), 4.30−4.27 (m, 1 H), 4.25−4.22 (m, 1 H), 1.32 (s,
9 H). MS m/z = 429.0 [M + H]+. Calcd for C25H22FN4O2: 429.2.
(S)-7-(2-Fluoropyridin-3-yl)-3-(3-methoxy-3-methylbut-1-yn-1-
yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (40).
A vial was charged with (S)-3-bromo-7-(2-fluoropyridin-3-yl)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (169 mg, 0.396
mmol), CuI (7.53 mg, 0.040 mmol), and Pd(Ph3P)4 (45.7 mg, 0.040
mmol). DMF (791 μL), i-Pr2NH (0.8 mL), and 3-methoxy-3-
methylbut-1-yne (143 μL, 1.187 mmol) were added in sequence, and
the vial was sealed and heated to 90 °C for 3.5 h. The reaction mixture
was diluted with water and extracted with EtOAc (2×). The combined
organic extracts were dried over sodium sulfate, filtered, and
concentrated under vacuum. The residue was purified by chromatog-
raphy on silica gel (0−100% of a 90:10:1 mix of DCM/MeOH/
NH4OH in DCM) to give a yellow oil. This oil was dissolved in
MeOH and loaded onto a 5-g SCX-2 acidic ion exchange column. The
column was eluted with methanol, then with 2N ammonia in
methanol. The basic fraction was concentrated under vacuum to give
(S)-7-(2-fluoropyridin-3-yl)-3-(3-methoxy-3-methylbut-1-ynyl)-5′H-
spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (167.79 mg,
0.378 mmol, 95% yield) as a white solid. 1H NMR (400 MHz,
DMSO-d6) δ = 8.38 (d, J = 2.2 Hz, 1 H), 8.25 (td, J = 1.5, 3.3 Hz, 1
H), 8.17−8.08 (m, 1 H), 7.76 (d, J = 2.2 Hz, 1 H), 7.64 (td, J = 1.7, 8.6
Preparation of Trimethyl((3-methyloxetan-3-yl)ethynyl)silane for
the Synthesis of 42. Step 1: Pyridinium chlorochromate (10.13 g, 47.0
mmol) and silica gel (10 g) were combined and ground with a mortar
and pestle until a fine, light-orange powder resulted. The powder was
transferred to a 500 mL round-bottom flask and suspended in DCM
(157 mL). 3-Methyl-3-oxetanemethanol (3.87 mL, 39.2 mmol) was
added in one portion via syringe, and the resulting mixture was stirred
for 24 h. The reaction the mixture was filtered through a pad of silica
gel, and the filter pad was washed with DCM (2×). The filtrate was
carefully evaporated, but not to dryness, to give 5.8193 g of a greenish
solution that corresponded to 32 wt % of the 3-methyloxetane-3-
1
carbaldehyde in DCM (as determined by H NMR analysis). This
1
solution was used directly in the next reaction. H NMR (400 MHz,
chloroform-d) δ = 9.96 (s, 1 H), 4.88 (d, J = 6.3 Hz, 2 H), 4.51 (d, J =
6.4 Hz, 2 H), 1.49 (s, 3 H).
Step 2: A 500 mL round-bottom flask was charged with
triphenylphosphine (19.51 g, 74.4 mmol) and DCM (93 mL) to
give a clear solution. The flask was cooled in an ice bath for 10 min,
and then carbon tetrabromide (12.34 g, 37.2 mmol) was added in one
portion. After 30 min of stirring, 3-methyloxetane-3-carbaldehyde
(5.8193 g, 32 wt % in DCM, 18.60 mmol) was added dropwise to the
thick, orange mixture via a pipet (with a 5 mL DCM flask-wash). The
resulting mixture was slowly warmed to room temperature over 1 h,
then was diluted with hexane (300 mL). The mixture was stirred for
10 min, resulting in the formation of a precipitate. The supernatant
was decanted onto a pad of silica gel and filtered. The remaining solid
was washed with 10% EtOAc/hexane (3 × 100 mL), each time
filtering through the silica gel pad. The combined filtrates were
evaporated to give 3-(2,2-dibromovinyl)-3-methyloxetane (3.539 g,
13.83 mmol, 74.3% yield) as a clear oil. 1H NMR (400 MHz,
chloroform-d) δ = 6.70 (s, 1 H), 4.82 (d, J = 5.8 Hz, 2 H), 4.39 (d, J =
6.1 Hz, 2 H), 1.61 (s, 3 H).
Step 3: A 250 mL round-bottom flask was charged with 3-(2,2-
dibromovinyl)-3-methyloxetane (2.177 g, 8.51 mmol) in THF (85
mL) to give a clear solution. The flask was cooled in a dry ice/acetone
bath for 15 min, and then n-butyllithium (7.83 mL, 19.56 mmol) (as a
2.5 M solution in hexane) was added dropwise over 5 min. The
resulting mixture was stirred for 45 min, and then trimethylchlor-
osilane (3.81 mL, 29.8 mmol) was added dropwise. The cooling bath
was removed, and the mixture was stirred at room temperature for 30
min. The reaction mixture was diluted with water (50 mL) and ether
(50 mL). The layers were separated, and the aqueous layer was
extracted with ether (25 mL). The combined organic extracts were
dried over magnesium sulfate, filtered, and evaporated. The residue
R
dx.doi.org/10.1021/jm5012676 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
was dissolved in a small amount of ether and filtered through a silica
gel pad. The filtrate was evaporated to give trimethyl((3-methyloxetan-
3-yl)ethynyl)silane (1.344 g, 7.99 mmol, 94% yield) as a pale-yellow
oil. 1H NMR (400 MHz, DMSO-d6) δ = 5.75 (s, 1 H), 4.61 (d, J = 5.3
Hz, 2 H), 4.33 (d, J = 5.6 Hz, 3 H), 0.14 (s, 9 H).
REFERENCES
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(S)-7-(2-Fluoropyridin-3-yl)-3-((3-methyloxetan-3-yl)ethynyl)-
5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine (42). A
round-bottom flask was charged with (S)-3-bromo-7-(2-fluoropyr-
idin-3-yl)-5′H-spiro[chromeno[2,3-b]pyridine-5,4′-oxazol]-2′-amine
(5.04 g, 11.80 mmol), copper(i) iodide (0.112 g, 0.590 mmol),
Pd(Ph3P)4 (0.682 g, 0.590 mmol), and tetrabutylammonium fluoride
trihydrate (4.63 g, 17.70 mmol). The flask was flushed with Ar(g), and
then THF (23.60 mL) and trimethyl((3-methyloxetan-3-yl)ethynyl)-
silane (3.35 mL, 17.70 mmol) were added in sequence. The flask was
fitted with a reflux condenser and heated to 70 °C for 2 h. The mixture
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with the aid of EtOAc (25 mL). The organic solution was washed with
water (2 × 25 mL), and the aquoeus washes were extracted with
EtOAc (25 mL). The organic extracts were combined, and the
combined solution was washed with brine (30 mL), dried over sodium
sulfate, filtered, and evaporated. The residue was purified by
chromatography on silica gel (0−50% of a 90:10:1 mix of DCM/
MeOH/NH4OH in DCM) to give to (S)-7-(2-fluoropyridin-3-yl)-3-
((3-methyloxetan-3-yl)ethynyl)-5′H-spiro[chromeno[2,3-b]pyridine-
5,4′-oxazol]-2′-amine (4.655 g, 10.52 mmol, 89% yield) as an off-white
solid. 1H NMR (400 MHz, DMSO-d6) δ = 8.36 (d, J = 2.2 Hz, 1 H),
8.25 (td, J = 1.6, 4.8 Hz, 1 H), 8.12 (ddd, J = 1.9, 7.5, 10.4 Hz, 1 H),
7.76 (d, J = 2.2 Hz, 1 H), 7.66−7.61 (m, 1 H), 7.57 (t, J = 1.8 Hz, 1
H), 7.49 (ddd, J = 1.9, 4.9, 7.4 Hz, 1 H), 7.39−7.35 (m, 1 H), 6.56 (s,
2 H), 4.78 (d, J = 5.5 Hz, 2 H), 4.45 (d, J = 5.6 Hz, 2 H), 4.32−4.28
(m, 1 H), 4.27−4.22 (m, 1 H), 1.66 (s, 3 H). MS m/z = 443.2 [M +
H]+. Calcd for C25H20FN4O3: 443.2.
ASSOCIATED CONTENT
■
S
* Supporting Information
General information, experimental data and characterization, X-
ray crystal structure information. This material is available free
of charge via the Internet at http://pubs.acs.org.
(4) Luo, X.; Yan, R. Inhibition of BACE1 for therapeutic use in
Alzheimer’s Disease. Int. J. Clin. Exp. Pathol. 2010, 3, 618−628.
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Maloney, J.; Hoyte, K.; Gustafson, A.; Liu, Y.; Lu, Y.; Bhangale, T.;
Graham, R. B.; Huttenlocher, J.; Bjornsdottir, G.; Andreassen, O. A.;
Accession Codes
The PDB accession code for the X-ray cocrystal of BACE1 +
compound 42 is 4RCD.
Jonsson, E. G.; Palotie, A.; Behrens, T. W.; Magnusson, O. T.; Kong,
̈
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 617-444-5352. Fax: 617-621-3907. E-mail: tdineen@
amgen.com. Address: Department of Medicinal Chemistry,
Amgen Inc., 360 Binney Street, Cambridge, Massachusetts,
United States.
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Present Address
#For V.F.P.: Sanofi, 153 Second Avenue, Waltham, Massachu-
setts 02451, United States.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; Aβ, β-amyloid; HEA, hydroxythethyl
amine; APP, amyloid precursor protein; BACE, β-site APP
cleaving enzyme; Pgp, P-glycoprotein
S
dx.doi.org/10.1021/jm5012676 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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