A Selective Prostaglandin E2 Receptor Subtype 2 (EP2) Antagonist Increases the Macrophage-Mediated Clearance of Amyloid-Beta Plaques

Article

pubs.acs.org/jmc

A Selective Prostaglandin E₂Receptor Subtype 2 (EP2) Antagonist

Increases the Macrophage-Mediated Clearance of Amyloid-Beta

Plaques

Brian M. Fox,*^,†Hilary P. Beck,^†Philip M. Roveto,^†Frank Kayser,^†Qingwen Cheng,^†Hannah Dou,^†

Toni Williamson,^‡James Treanor,^†Hantao Liu,^‡Lixia Jin,^†Guifen Xu,^†Ji Ma,^†Songli Wang,^†

and Steven H. Olson^†

^†Amgen South San Francisco, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States

^‡Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States

S

* Supporting Information

ABSTRACT: A high-throughput screen resulted in the dis-

covery of benzoxazepine 1, an EP2 antagonist possessing low

microsomal stability and potent CYP3A4 inhibition. Modular

optimization of lead compound 1 resulted in the discovery of

benzoxazepine 52, a molecule with single-digit nM binding

aﬃnity for the EP2 receptor and signiﬁcantly improved micro-

somal stability. It was devoid of CYP inhibition and was ∼4000-

fold selective against the other EP receptors. Compound 52 was

shown to have good PK properties in CD-1 mice and high CNS permeability in C57Bl/6s mice and Sprague−Dawley rats. In an

ex vivo assay, it demonstrated the ability to increase the macrophage-mediated clearance of amyloid-beta plaques from brain

slices in a dose-dependent manner.

A similar phenotype has been demonstrated in AD patients

where microglia lose their ability to clear Aβ plaques and

develop a consistent proinﬂammatory response.^22,23Thus, it

has been hypothesized that microglia are unable to clear,

through phagocytosis, the signiﬁcant amount of aggregated Aβ

present in the brains of AD patients. This results in a chronic

Aβ-stimulated microglial-mediated inﬂammatory response that

causes neurotoxicity and exacerbates the disease pathology.^20,21,24

Although many factors may play a role in the accumulation

of Aβ in Alzheimer’s disease, several lines of evidence indicate

that prostaglandin E₂(PGE₂) is a key contributor. PGE₂dose-

dependently inhibits macrophage-mediated phagocytosis,²⁵

and elevated levels of PGE₂have been observed in the

brains of patients with AD.²⁶PGE₂is the natural ligand of four

prostanoid receptors, termed prostaglandin E₂receptor sub-

types 1−4 (EP1, EP2, EP3, and EP4).²⁷Several lines of evidence

indicate that PGE₂inhibits the microglial-mediated phagocytosis

of aggregated Aβ through the EP2 receptor. Microglia from mice

lacking the EP2 receptor (EP2−/−) have signiﬁcantly increased

phagocytosis of aggregated Aβ compared to wild-type (wt)

microglia in vitro.³Microglia lacking EP2 also demonstrated

enhanced phagocytosis of Aβ plaques present on hippocampal

sections from patients who died with Alzheimer’s disease.³

Deletion of the EP2 receptor in the APPSwe-PS1ΔE9 mouse

model of familial Alzheimer’s disease resulted in a signiﬁcant

reduction of Aβ plaques and Aβ40 and Aβ42 levels compared

INTRODUCTION

■

Alzheimer’s disease (AD) is characterized by the accumulation

of amyloid-beta (Aβ) plaques, neuroﬁbrillary tangles, and

neurodegeneration.¹Although the exact cause of neuro-

degeneration is still under debate, it is likely that Aβ plaques

contribute because aggregated Aβ is directly toxic to neurons.^2,3

Several lines of evidence indicate that microglia are responsible

for the clearance of Aβ plaques in the CNS. Microglia surround

and inﬁltrate Aβ plaques in human and mouse brain.⁴⁻¹¹

Microglia clear Aβ plaques by phagocytosis,^2,4,12−14and

activation of microglia by Aβ immunization or LPS exposure

causes an increase in microglial-mediated phagocytosis.^11,13,15

While clearance of Aβ plaques should be beneﬁcial, exposure of

microglia to Aβ plaques elicits an innate immune response that

causes the release of proinﬂammatory cytokines such as IL-1β,

IL-6, tumor necrosis factor α (TNFα), the chemokines IL-8,

MIP-1α, and MCP-1, the growth factor (M-CSF), along with

glutamate, complement proteins, peroxynitrite, and superoxide

radicals.¹⁶⁻¹⁹This potent inﬂammatory response results in

oxidative damage and neurotoxicity.

Recent evidence indicates that although microglia are

beneﬁcial in clearing plaques at the onset of AD, they become

dysfunctional and lose their ability to clear plaques as the

disease progresses. Chronic exposure of microglia to aggregated

Aβ in mice results in the reduced expression of Aβ-binding

receptors and Aβ-degrading enzymes, resulting in a reduction

in their ability to clear plaques by phagocytosis.^20,21While there

is a reduction in the ability to clear plaques, the microglia con-

tinue to produce a robust neurotoxic inﬂammatory response.

Received: April 10, 2015

A

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

to mice possessing the EP2 receptor.²⁸The decrease in Aβ was

shown to be mediated by microglia by demonstrating that

APPSwe-PS1ΔE9 mice that were irradiated and then implanted

with bone marrow from EP2−/− mice had reduced Aβ levels

compared to mice implanted with bone marrow from wt mice.²⁹

In addition to playing a role in Aβ phagocytosis, the EP2

receptor is required to elicit the microglial-mediated innate

immune response. Deletion of EP2 signiﬁcantly reduces

inﬂammation and the resulting oxidative damage after a LPS

challenge.^28,30,31Exposure of neurons to LPS causes neuro-

toxicity as a result of the microglial-mediated activation of

innate immunity that is EP2 dependent.³²In a similar manner,

incubation of primary neurons with aggregated Aβ causes

neurotoxicity that is exacerbated by the presence of wt

microglia. In contrast, incubation of primary neurons with

aggregated Aβ in the presence of EP2−/− microglia caused no

neurotoxicity.³While this data clearly implicated the involve-

ment of the EP2 receptor in the phagocytosis of Aβ plaques

and the resulting neurotoxic inﬂammatory response, the role

of EP2 in the development of Alzheimer’s disease remained

unclear. Recent evidence demonstrated that aging or Aβ

accumulation caused the upregulation of microglial EP2

signaling. This increased proinﬂammatory gene expression

and suppressed beneﬁcial chemokine production and chemo-

taxis, resulting in a reduction in Aβ clearance.³³This data may

explain why microglia become dysfunctional in patients with

AD and are unable to clear accumulating Aβ plaques.

The EP2 receptor has been implicated in the etiology of

several neurodegenerative diseases in addition to Alzheimer’s

disease, including Parkinson’s disease (PD), amyotrophic lateral

sclerosis (ALS), and epilepsy.³⁴Furthermore, deletion of the

EP2 receptor has demonstrated beneﬁcial eﬀects in mouse

models of AD, PD, and ALS.³⁴For many years, the lack of

selective tool molecules have made it diﬃcult to further interro-

gate the role of the EP2 receptor in these neurodegenerative

diseases, however, recent reports describe the discovery of

selective EP2 antagonists,³⁵⁻⁴¹including the demonstration

that they reduce brain inﬂammation and neuronal injury in a

mouse model of status epilepticus.^37,38

Table 1. Potency, Selectivity, and in Vitro ADME Data for 1

a

EP2 Potency

hEP2 SPA IC₅₀: 0.07 0.04 μM

rEP2 SPA IC₅₀: 0.1 0.05 μM

mEP2 SPA IC₅₀: 0.1 0.03 μM

hEP2 cAMP IC₅₀: 0.6 0.4 μM

rEP2 cAMP IC₅₀: 0.3 0.2 μM

mEP2 cAMP IC₅₀: 0.06 0.03 μM

a

Selectivity

hEP1 SPA IC₅₀: >30 μM

hEP3 SPA IC₅₀: 23 10 μM

hEP4 SPA IC₅₀: 3.5 1.6 μM

In Vitro ADME

CYP3A4_inh@ 3 μM = 97%

HLM CL: >399 μL/min·mg

CYP2D6_inh@ 3 μM = 48%

RLM CL: >399 μL/min·mg

a

Data are reported as the average of a minimum of two

determinations.

Lead compound 1 was separated into fragments consisting of

the benzothiophene ring, pyridine ring, and benzoxazepine

core, and each was optimized independently. The potent inhibi-

tion of CYP3A4 and 2D6 by 1 is of concern because these two

isoforms metabolize more than 50% of clinically important

drugs; therefore, inhibition of either of these isoforms has the

potential to cause signiﬁcant drug−drug interactions in patients

receiving more than one drug substance.⁴²The pyridine ring

was suspected to be responsible for the potent CYP inhibition

observed for benzoxazepine 1 due to the well-documented

ability of pyridine nitrogens to interact with the iron in the

heme-containing CYP enzymes.⁴³The inhibition of CYP

enzymes by pyridines can be mitigated by the introduction of

steric encumberance around the pyridine nitrogen or by

reducing the basicity of the pyridine nitrogen.⁴³Moving the

pyridyl nitrogen to the 2-position resulted in a signiﬁcant

decrease in the inhibition of both CYP3A4 and 2D6, likely

due to the increased steric bulk around the pyridyl nitrogen

(Table 2). Unfortunately, this also resulted in a 10-fold

decrease in EP2 potency. The 4-pyridyl analogue 3 had similar

CYP inhibition values compared to 1 and resulted in a 3-fold

loss in EP2 potency. Reducing the basicity of the pyridine

nitrogen by replacement with pyrimidine 4 resulted in an

increase in EP2 potency but no improvement in CYP3A4

inhibition. On the basis of these results, molecules were pre-

pared that had both decreased basicity and increased steric bulk

around the pyridine nitrogen. This provided methoxy pyridines

5 and 6 and pyridone 7 that were very successful in reducing

the CYP3A4 inhibition while retaining EP2 potency.

The second approach investigated to reduce the CYP inhibi-

tion was to remove the pyridine nitrogen altogether. Con-

verting the pyridine ring to a phenyl ring provided analogue 8

that was devoid of CYP3A4 inhibition but had signiﬁcantly

reduced EP2 potency. Variation of substituents on the phenyl

ring revealed that hydrophobic groups were not tolerated

(chloro and methyl analogues 9, 10, and 15). In contrast,

placing H-bond acceptors at the 3- and 4-positions provided

compounds 11−14 and 16 that possessed increased EP2

potency compared to phenyl analogue 8. Two of the most

potent phenyl analogues prepared were sulfone 14 and nitrile

16, each being 100-fold more potent than phenyl analogue 8. In

addition, they possessed signiﬁcantly reduced CYP inhibition

Consideration of the data discussed above indicates that

inhibiting the activation of the microglial EP2 receptor with a

small molecule antagonist provides an extremely attractive

approach to treating AD by increasing the phagocytosis of

neurotoxic Aβ plaques while at the same time abrogating the

neurotoxic inﬂammatory response caused when microglia are

exposed to aggregated Aβ.

RESULTS AND DISCUSSION

■

A high-throughput screen of our small molecule library resulted

in the discovery of benzoxazepine 1 as a novel EP2 antagonist

with moderate potency across the human, rat, and mouse

receptors (Table 1). Compound 1 was greater than 400-fold

selective over the EP1 receptor, 300-fold selective over the EP3

receptor, and 50-fold selective against the EP4 receptor.

Benzoxazepine 1 was a potent inhibitor of both CYP3A4 and

2D6 and had low microsomal stability in human and rat liver

microsomes. Despite these issues, we considered compound 1

to be a good starting point for lead optimization due in part to

the low MW (402.5 g/mol), low PSA (34.5 Å²), absence of

H-bond donors, and reasonable cLogD_7.4(5.3) that should

provide 1, and compounds of similar structure, appreciable

CNS permeability.

B

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 2. hEP2 IC₅₀Values and CYP Inhibition Data for Compounds 1−16

a

Data with SD are reported as the average of a minimum of two determinations.

values compared to lead compound 1 while having similar EP2

IC₅₀values. Investigating analogues of the pyridine ring

revealed that the pyridine nitrogen was responsible for

CYP3A4 inhibition and that H-bond acceptors greatly

C

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

improved EP2 potency. While the SAR trend for the inhibition

of CYP2D6 was less straightforward, several replacements

of the pyridine ring, such as the pyridone ring in 7 and the

4-methylsulfonylphenyl ring in 14, provided analogues that

possessed similar EP2 potency compared to 1 while also

signiﬁcantly decreasing CYP3A4 and CYP2D6 inhibition.

Incubation of lead compound 1 with liver microsomes

revealed that multiple oxidative metabolites were formed at

several positions of this molecule. This included several

oxidative metabolites of the benzothiopene ring of 1, therefore,

replacement of this ring was a high priority. Initial work

indicated that maintaining a hydrophobic group in the area

occupied by the benzothiophene phenyl ring was crucial to

maintaining EP2 potency. For example, removing the phenyl

ring to provide thiophene 17 resulted in complete loss of EP2

potency while altering the position of the phenyl ring to the

2-linked benzothiopene 18 resulted in a compound with

micromolar potency (Table 3). The importance of the phenyl

ring was also demonstrated by the signiﬁcant increase in

potency of naphthalene 20 compared to phenyl analogue 19.

Attempts to replace the second phenyl ring of naphthalene

analogue 20 with monosubstituted phenyl analogues of 19

indicated that polar groups were not tolerated (data not

shown). In contrast, m-methyl analogue 22 was equipotent to

19, while the o- and p-methyl isomers 21 and 23 were devoid of

EP2 activity. The meta position was also optimal for the

chlorine analogues with m-chloro derivative 25, demonstrating

a 20-fold increase in potency over 19 while the o- and p-chloro

isomers 24 and 26 were equipotent to 19. Despite the

improved potency, m-chlorophenyl analogue 25 was still

10-fold less potent than compound 1, which possessed a

benzothiophene ring. This led to the conclusion that

heterocyclic rings containing a phenyl ring in a similar position

to the benzothiophene phenyl ring should be explored.

It quickly became clear that the SAR was very sensitive to

thiophene replacements with increased polarity. Incorporation

of a nitrogen atom to provide benzoisothiazole 27 resulted in

a 6-fold loss in EP2 potency while increasing the polarity

further by converting this ring to a benzimidazole 28 resulted

in nearly a 10-fold loss in potency compared to compound 1.

Results obtained from 1-linked indole 29 and 3-linked indole

analogues 30 and 31 further demonstrated that sulfur and carbon

atoms were preferred at positions analogous to the 1- and

2-positions of the benzothiophene ring.

The 1-linked indole ring contained in 29 proved to be the

most potent heterocyclic ring replacement for the benzothio-

phene in this series, but it was still more than 4-fold less potent

than lead compound 1. Earlier work investigating the SAR of

phenyl analogue 19 demonstrated a strong preference for

hydrophobic groups in this area of the binding pocket with a

chlorine atom, providing the largest increase in EP2 potency.

Therefore, in an attempt to increase the potency of the 1-linked

indole series, several chloroindole isomers were prepared.

Within this series, 3-chloroindole 32 and 5-chloroindole 34 had

the highest EP2 binding aﬃnity, with IC₅₀values of 60 and

80 nM, respectively, while 4-, 6-, and 7-substituted indole

analogues 33, 35, and 36 all had signiﬁcantly higher IC₅₀values.

Furthermore, the 3-, 4-, and 6-chloroindole isomers 32, 33,

and 35 were all shown to be weak partial agonists for the

human EP2 receptor (data not shown). These data warranted

further investigation of 5-substituted indole analogues. The

5-cyanoindole, 5-ﬂuoroindole, and 5-chloroindoline com-

pounds 37, 38, and 39 all demonstrated EP2 aﬃnity in the

SPA assay that was comparable to 1, while 38 and 39 also

demonstrated a 10-fold increase in potency in the cAMP assay

compared to benzothiophene 1. The microsomal stability of

each compound in Table 3 was similar to benzothiophene 1.

The lack of improvement in microsomal stability is not un-

expected, as the metabolite identiﬁcation of lead compound 1

indicated that metabolism occurred at several positions of this

molecule. It is likely that reducing metabolism in a single

section of this molecule will not result in a detectable diﬀerence

in microsomal stability but will require several changes in the

overall structure.

In addition to oxidation of the benzothiophene ring,

metabolite identiﬁcation indicated that the methoxy group on

the benzoxazepine ring is readily demethylated when 1 is

incubated with liver microsomes. Thus, it is likely that removal

or replacement of the methoxy group would be required to

provide a molecule with acceptable metabolic stability. Re-

moving the methoxy group to provide 40 resulted in a 15-fold

reduction in EP2 potency. The phenol metabolite 41 was also

prepared and shown to be greater than 20-fold less active

against the EP2 receptor. Increasing the size of the methyl ether

to ethyl 42 and isopropyl 43 also resulted in a signiﬁcant loss in

potency. Replacement of the methyl ether with a methyl group

provided analogue 44 that showed similar potency compared to

1. Chlorine proved to be the optimal replacement of the methyl

ether, providing compound 45 that was equipotent to 1 while

at the same time removing a metabolic liability that contributed

to the poor microsomal stability of the series. Not surprisingly,

because of the multiple metabolic soft spots none of the com-

pounds in Table 4 demonstrated an improvement in micro-

somal stability compared to methyl ether 1.

Changes to the oxazepine ring were also investigated

(Table 5). Replacing the oxygen atom of the oxazepine ring

with a carbon atom provided a compound with similar potency

(benzoazepine 46 vs benzoxazepine 40), while replacement

with a nitrogen atom resulted in a minimal loss in potency (47

and 48 vs 40). Both lactam analogues prepared (49 and 50)

demonstrated low EP2 aﬃnity.

The modular approach taken to investigate the SAR of

benzoxazepine 1 resulted in several key discoveries. Replace-

ment of the pyridine ring provided several compounds that

retained EP2 potency and were devoid of CYP3A4 inhibition,

including methoxy pyridines 5 and 6, pyridone 7, 3-

cyanophenyl analogue 16, and 4-methylsulfonylphenyl ana-

logue 14. Replacement of the benzothiophene ring with a 5-

ﬂuoroindole ring 38 or a 5-chloroindoline ring 39 provided

analogues that were equipotent to 1 in the SPA assay and

signiﬁcantly more potent in the cAMP assay. It was discovered

that replacement of the oxazepine oxygen atom with a carbon

atom resulted in a compound with similar EP2 potency (46 vs 40).

Furthermore, the methyl ether could be replaced with a

chlorine atom with no loss in potency in the SPA assay and a

minimal loss of activity in the cAMP assay. Because of the

multiple sites of metabolism, no single change made during this

modular approach resulted in an improvement in microsomal

stability. However, it is likely that many of these changes have

an impact on the microsomal turnover that is undetectable

because the groups that remain are metabolically labile. In this

case, it is predicted that the combination of several groups

would provide a molecule with a detectable improvement in

microsomal stability. In a similar manner, the combination of

groups that provided modest increases in potency would be

D

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 3. hEP2 IC₅₀Values for Compounds 17−39

a

Data with SD are reported as the average of a minimum of two determinations.

additive and provide compounds with signiﬁcant increases in

EP2 potency.

The results obtained from combining some of the best frag-

ments are shown in Figure 1. As discussed earlier, replacement

of the methyl ether of 1 with chlorine while retaining the

benzothiophene and pyridine ring to provide 45 resulted in

no improvement in microsomal stability. However, when the

methyl ether to chlorine change was coupled with the

E

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 4. hEP2 IC₅₀Values for Compounds 40−45

Table 5. hEP2 IC₅₀Values for Compounds 46−50

a

Data with SD are reported as the average of a minimum of two

determinations.

replacement of the pyridine ring by a pyridone ring, a

signiﬁcant improvement in microsomal stability was achieved

(51 vs 1 and 45). Replacement of the benzothiophene ring in

51 with the 5-ﬂuoroindole ring provided compound 52 that

possessed similar microsomal stability compared to 51 and a

signiﬁcant increase in EP2 potency. Combination of the three

optimal replacements of benzothiophene to 5-ﬂuoroindole,

methyl ether to chlorine, and pyridine to pyridone provided

benzoxazepine 52 that was 10-fold more potent than 1 in the

SPA and cAMP assays, was devoid of CYP inhibition, and

possessed signiﬁcantly increased microsomal stability compared

to lead compound 1.

Lead compound 1 displayed greater than 400-fold selectivity

against the hEP1 receptor, 300-fold selectivity against the hEP3

receptor, and 50-fold selectivity against the hEP4 receptor.

Signaling through the EP receptors has versatile and often

opposing actions in many tissues.²⁷All four EP receptors are

expressed in the CNS including the expression of EP1 and EP2

on microglia.⁴⁴Because of the presence of multiple EP

receptors in the CNS and the wide array of physiological

roles mediated by them, one of the objectives of this project

was to increase the selectivity against the EP3 and EP4

receptors. This would ensure that any eﬀects observed would

be due to signaling through the EP2 receptor alone. Conversion

of the methyl ether of benzoxazepine 1 to chloride 45 failed

to provide any increase in selectivity (Figure 2). Combining

this change with the conversion of the pyridine ring to a

pyridone ring provided 51 that was 900-fold selective against

the hEP4 receptor. Replacing the benzothiophene ring with a

5-ﬂuoroindole ring (52) or adding a methyl group alpha to the

oxazepine nitrogen (53) further increased the selectivity to

∼4000-fold. Finally, combination of the four changes discussed

above provided benzoxazepine 54 that was shown to have a

10000-fold higher binding aﬃnity for the hEP2 receptor

compared to the hEP4 receptor. While it required several

changes in the structure of compound 1 to provide high

selectivity against the hEP4 receptor, each of the compounds

shown in Figure 2, with the exception of 1, had hEP3 binding

aﬃnity values greater than the highest tested concentration.

The lack of binding aﬃnity for the hEP1 receptor was also

maintained for all of the compounds in this series.

Benzoxazepine 52 demonstrated comparable potency across

the human, rat, and mouse EP2 receptors (Table 6). Com-

pound 52 was further proﬁled against the human prostanoid

receptors DP, TP, IP, and CRTH2. For each receptor, an

aequorin assay was used to determine an IC₅₀value of the

antagonism of an agonist ligand, as well as an EC₅₀value in the

absence of an added agonist. Compound 52 was greater than

660-fold selective in the hEP2 cAMP assay versus all four

receptors.

Benzoxazepine 52 had moderate clearance in CD-1 mice

with a corresponding half-life of 3.4 h and good bioavailability

and exposure when dosed orally (Table 7). The ability of com-

pound 52 to access the CNS was measured in both C57BL/6s

mice and Sprague−Dawley rats. Animals were dosed orally with

52 followed by sacriﬁcing mice after 2 h and rats after 3 h.

Concentrations measured in plasma and brain homogenate

demonstrated that compound 52 had good CNS exposure in

C57BL/6s and in Sprague−Dawley rats with brain/plasma

ratios of 0.7 and 0.9, respectively.

The ability of benzoxazepine 52 to increase macrophage-

mediated Aβ phagocytosis was studied using an ex vivo assay.

Plaque-bearing brain slices from 18 month old Tg2576 mice⁴⁵

were incubated for 24 h in the presence of mouse peritoneal

macrophage IC21 cells (ATCC TIB-186) and vehicle or

F

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Human EP2 IC₅₀values, microsomal stability, and CYP3A4 inhibition data for compounds 1, 45, 51, and 52. Data with SD are reported as

the average of a minimum of two determinations.

Figure 2. Human EP1, EP2, EP3, and EP4 IC₅₀values for compounds 1, 45, and 51−54. Data are reported as the average of a minimum of two

determinations.

Table 6. Potency and Selectivity Data for 52

Table 7. Pharmacokinetic Data for 52

a

EP2 Potency

CD-1 mouse PK (1.0 mg/kg iv)

CD-1 mouse PK (5.0 mg/kg po)

CL = 0.92 L/h/kg

t_1/2= 3.4 h

AUC = 5.7 μM·h

C_max= 1.2 μM

hEP2 SPA IC₅₀: 0.008 0.004 μM hEP2 cAMP IC₅₀: 0.05 0.04 μM

rEP2 SPA IC₅₀: 0.01 0.006 μM

rEP2 cAMP IC₅₀: 0.03 0.01 μM

V_dss= 2.6 L/kg

F = 44%

mEP2 SPA IC₅₀: 0.01 0.003 μM mEP2 cAMP IC₅₀: 0.004 0.001 μM

C57BL/6s mouse PK (200 mg/kg po)

SD rat PK (100 mg/kg po)

Selectivity

brain conc @ 2h = 11500 μg/L

plasma conc @ 2h = 16400 μg/L

brain conc @ 3h = 3440 μg/L

plasma conc @ 3h = 3920 μg/L

hDP Aeq IC₅₀: >33 μM

hIP Aeq IC₅₀: >33 μM

hTP Aeq IC₅₀: >33 μM

hCRTH2 Aeq IC₅₀: >33 μM

a

Data are reported as the average of a minimum of two

determinations.

the IC21-mediated phagocytosis of Aβ plaques present on brain

slices (Figure 3D). The image of a brain slice incubated with

1 μM 52 (Figure 3C) clearly demonstrates an increase in

Aβ phagocytosis compared to vehicle (Figure 3A), as shown by

the reduction in the number and size of plaques and the

internalization of Aβ by IC21 cells (green-labeled Aβ inside

compound 52. The results are shown in Figure 3, with mouse

IC21 cells labeled red and Aβ peptide labeled green. Incubation

with benzoxazepine 52 in a concentration range from 10 nM to

3.0 μM demonstrated a consistent dose-dependent increase in

G

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 3. Images from the in vitro phagocytosis assay showing the incubation of brain slices from 18 month old Tg2576 mice incubated with mouse

IC21 cells in the presence of (A) vehicle (DMSO), (B) 10 μg/mL Aβ antibody 2.1, (C) 1 μM compound 52. (D) Dose−response of 52.

a

Scheme 1

a

Reagents and conditions: (a) benzo[b]thiophen-3-ylboronic acid, K₃PO₄, XPhos, Pd(OAc)₂, n-BuOH, 100 °C, 83%; (b) ethylene glycol, p-TsOH,

toluene reﬂux, quant; (c) DEAD, PPh₃, THF rt, 65%; (d) (i) TFA, DCM, 91%, (ii) NaBH₄, MeOH, quant; (e) RCHO, NaBH(OAc)₃,

dichloroethane, 15−98%.

red-labeled IC21 cells). The phagocytosis induced by com-

pound 52 was similar to that caused by incubation with an Aβ

antibody (Figure 3B).

of the resulting cyclic imine using NaBH₄in MeOH to provide

benzoxazepine 59 in 91% yield over two steps. Finally, reduc-

tive amination using the corresponding aldehydes and NaBH-

(OAc)₃provided analogues 1−16.

Nucleophilic displacement of mesylate 61, prepared from

alcohol 60 by phenol 55 provided the Boc-protected amine 62

in 81% yield (Scheme 2). Deprotection of the amine with TFA

followed by reduction with NaBH₄provided benzoxazepine

63 in 91% yield over two steps. Reductive amination of amine

63 with nicotinaldehyde provided the penultimate intermediate

64 in moderate yield. Palladium-catalyzed or copper-catalyzed

coupling using bromide 64 directly or following conversion to

the corresponding boronic acid provided analogues 17−39.

CHEMISTRY

■

The preparation of analogues 1−16 began by introducing the

benzothiophene ring through a Suzuki coupling employing

Pd(OAc)₂and XPhos to provide 56 in 83% yield (Scheme 1).⁴⁶

Protection of the aldehyde using ethylene glycol provided cyclic

acetal 57 in quantitative yield followed by Mitsunobu reaction

to provide the Boc-protected amine 58 in 65% yield. Formation

of the oxazepine ring was accomplished by treating 58 with

triﬂuoroacetic acid in dichloromethane followed by reduction

H

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Scheme 2

a

Reagents and conditions: (a) MsCl, NEt₃, DCM, 0 °C−rt, quant; (b) 55, K₃PO₄, DMF, 75 °C, 81%; (c) (i)TFA, DCM, rt, quant, (ii) NaBH₄,

MeOH, 0 °C−rt, 91%; (d) nicotinaldehyde, NaBH(OAc)₃, DCM, rt, 45%; (e) RB(OH)₂, base, phosphine ligand, Pd catalyst, or (i) BuLi,

triisopropylborate, THF, −78 °C, (ii) HCl, (iii) RX, K₃PO₄, XPhos, Pd(OAc)₂, n-butanol, 100 °C, or R₂NH, K₃PO₄, CuI, (1S,2S)-(+)-N,N′-

dimethyl-1,2-cyclohexanediamine, dioxane, 110 °C.

a

Scheme 3

a

Reagents and conditions: (a) 61, K₂CO₃, DMF, rt−65 °C, 56−73%; (b) benzo[b]thiophen-3-ylboronic acid, Na₂CO₃, Pd(dppf)Cl₂·CH₂Cl₂, water,

DMF, 80 °C, 81−87%; (c) (i)TFA, DCM, rt, quant, (ii) NaBH₄, EtOH, rt, quant; (d) nicotinaldehyde, NaBH(OAc)₃, DCM, rt, 50−67%.

a

Scheme 4

a

Reagents and conditions: (a) (i) AlCl₃, octane-1-thiol, DCM rt, (ii) nicotinaldehyde, NaBH₃CN, 3 Å molecular sieves, MeOH, rt, 46%; (b)

benzo[b]thiophen-3-ylboronic acid, Pd(OAc)₂, XPhos, K₃PO₄, n-butanol, 100 °C, 69%; (c) bromoethane or 2-bromopropane, NaH, DMF, rt, 9−44%.

The synthesis of analogues that replace the methoxy group of

1 with hydrogen 40 or methyl 44 was accomplished using

similar chemistry as that used to prepare analogues 1−39 and is

shown in Scheme 3. Nucleophilic addition of phenols 65 and

66 to mesylate 61 provided the Boc-protected amines 67 and

68 in good yield. Suzuki coupling with benzo[b]thiophen-3-

ylboronic acid provided compounds 69 and 70 in 87% and 81%

yield, respectively. Deprotection of the amines with TFA

followed by reduction of the resulting imines using NaBH₄in

ethanol provided benzoxazepine 71 and 72 in quantitative

yield. Reductive amination with nicotinaldehyde under stan-

dard conditions provided 40 and 44 in 50% and 67% yield,

respectively.

Phenol 73 was prepared by demethylation of intermediate 63

using AlCl₃and octanethiol followed by reductive amination

with nicotinaldehyde (Scheme 4).⁴⁷Suzuki coupling of 73 with

benzo[b]thiophen-3-ylboronic acid provided phenol 41 in 69%

yield. Alkylation of phenol 41 with ethyl bromide or isopropyl

bromide using NaH in DMF provided ethyl ether 42 and

isopropyl ether 43.

Synthesis of chloride analogue 45 began by formylation of

4-bromo-2-chlorophenol 74 using the Duﬀ reaction to provide

75 in 96% yield (Scheme 5). Protection of the aldehyde as the

cyclic acetal followed by Mitsunobu reaction provided 77 in

65% yield. Suzuki coupling followed by deprotection of the

amine and NaBH₄reduction of the cyclic imine provided

I

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Scheme 5

a

Reagents and conditions: (a) hexamethylenetetramine, TFA, 90 °C, 96%; (b) ethylene glycol, p-TsOH, benzene reﬂux, 65%; (c) 60, DEAD, PPh₃,

THF, rt, 75%; (d) benzo[b]thiophen-3-ylboronic acid, Pd(OAc)₂, SPhos, K₃PO₄, n-butanol, 70 °C, 84%; (e) (i) TFA, DCM, rt, 62%, (ii) NaBH₄,

MeOH, rt, 68%; (f) nicotinaldehyde, NaBH(OAc)₃, DCM, rt, 45%.

a

Scheme 6

a

Reagents and conditions: (a) benzo[b]thiophen-3-ylboronic acid, Pd(OAc)₂, XPhos, K₃PO₄, n-butanol, 100 °C, 88%; (b) NaN₃, TFA, HCl, THF,

rt, 12%; (c) LAH, THF, 0 °C−rt, 50%; (d) nicotinaldehyde, NaBH(OAc)₃, DCM, rt, 44%.

a

Scheme 7

a

Reagents and conditions: (a) benzo[b]thiophen-3-ylboronic acid, Pd(OAc)₂, XPhos, K₃PO₄, n-butanol, 100 °C, 51%; (b) nicotinaldehyde,

NaBH(OAc)₃, DCM, rt, 53%; (c) formaldehyde, NaBH₃CN, 3 Å molecular sieves, MeOH, 33%.

benzoxazepine 79. Reductive amination with nicotinaldehyde

provided chloride analogue 45 in moderate yield.

aniline nitrogen of 47 by reductive amination with form-

aldehyde.

Suzuki coupling of bromide 80 with benzo[b]thiophen-3-

ylboronic acid provided 81 in 88% yield (Scheme 6). Ring

expansion using sodium azide⁴⁸followed by reduction of

the resulting lactam with LAH provided benzoazepine 83.

Reductive amination with nicotinaldehyde gave benzoazepine

core analogue 46 in moderate yield.

Preparation of benzodiazepine analogues 47 and 48 began

with the Suzuki coupling of commercially available chloride

84 (Scheme 7). Reductive amination using nicotinaldehyde

provided core analogue 47 in 53% yield. Methylated

analogue 48 was obtained through the methylation of the

Alkylation of phenol 55 with ethyl bromoacetate provided

ester 86 in excellent yield (Scheme 8). Introduction of the

benzothiophene ring was accomplished using the standard

Suzuki coupling procedure to provide 87 in 84% yield. The ﬁnal

step was achieved in a stepwise fashion by ﬁrst forming the

benzylamine intermediate through a reductive amination with

pyridine-3-ylmethanamine at rt, followed by heating at reﬂux to

eﬀect ring closure and provide benzoxazapinone 49 in 73% yield.

The preparation of benzoxazapinone 50 began by oxidizing

the aldehyde of intermediate 62 with sodium chlorite followed

by methylation of the resulting acid with TMS−diazomethane

J

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Scheme 8

a

Reagents and conditions: (a) ethyl bromoacetate, K₂CO₃, DMF rt, 91%; (b) benzo[b]thiophen-3-ylboronic acid, Pd(dppf)Cl₂·CH₂Cl₂, Na₂CO₃,

water, DMF, 70 °C, 84%; (c) (i) pyridine-3-ylmethanamine, NaBH(OAc)₃, dichloroethane, rt, (ii) reﬂux, 73%.

a

Scheme 9

a

Reagents and conditions: (a) (i) NaClO₂, sulfamic acid, dioxane, water, rt, 43%, (ii) TMSCHN₂, MeOH, DCM, 0 °C−rt, 46%; (b) (i) TFA,

DCM, rt, (ii) DIPEA, MeOH, 65 °C, 52%; (c) benzo[b]thiophen-3-ylboronic acid, Pd(OAc)₂, XPhos, K₃PO₄, n-butanol, 100 °C, 57%;

(d) 3-(bromomethyl)pyridine hydrobromide, NaH, THF, rt, 28%.

to yield methyl ester 88 (Scheme 9). Formation of the ring was

accomplished by deprotection of the amine with TFA followed

by cyclization under basic conditions. The benzothiophene ring

was introduced via Suzuki coupling to provide the penultimate

intermediate 90 in 57% yield. Alkylation of the lactam nitro-

gen was accomplished using NaH as the base providing core

analogue 50.

The synthesis of analogues 51−54 is shown in Scheme 10.

Mitsunobu reactions employing the appropriate alcohols

provided amines 77 with R = H and 91 with R = methyl.

Treatment of the amines with triﬂuoroacetic acid in dichloro-

methane aﬀorded imines 92 and 93 in quantitative yield.

Benzothiophene analogue 94 was prepared by introducing the

benzothiophene ring with a Suzuki coupling ﬁrst, followed by

reduction of the cyclic imine with NaBH₄in MeOH. In

contrast, indole analogues 95 and 96 were prepared by

reducing the imine with NaBH₄ﬁrst, followed by introduction

of the 5-ﬂuoroidole ring through a copper-catalyzed coupling.

Finally, reductive amination of amines 79 (from Scheme 7) and

94−96 with 2-hydroxyisonicotinaldehyde provided analogues

51−54 in good to moderate yield.

replacements for the benzothiophene ring, pyridine ring, and

methyl ether of 1 that addressed these liabilities. Combination

of these replacements provided benzoxazepine 52 possessing

8 nM binding aﬃnity for the hEP2 receptor and a 50 nM IC₅₀

value in the hEP2 cAMP assay. The improvement in hEP2

potency was accomplished while maintaining a similar

molecular weight compared to 1 and lowering the cLogD_7.4

by more than 2 orders of magnitude (cLogD_7.4of 5.26 vs 2.79).

Compound 52 was devoid of CYP inhibition at 3 μM, had

∼4000-fold higher binding aﬃnity for the hEP2 receptor

compared to the hEP1, hEP3, and hEP4 receptors, and was

greater than 660-fold more potent in the hEP2 cAMP assay

compared to functional assays for the human DP, TP, IP, and

CRTH2 receptors. Benzoxazepine 52 was shown to have

moderate clearance and good exposure in CD-1 mice and good

CNS exposure in both C57Bl/6s mice and Sprague−Dawley

rats. Finally, 52 demonstrated the ability to increase the

macrophage-mediated phagocytosis of Aβ plaques present on

mouse brain slices. To our knowledge, this is the ﬁrst example

of an EP2 antagonist eliciting an increase in Aβ phagocytosis in

a similar manner to that caused by EP2 knockout microglia.

CONCLUSION

■

EXPERIMENTAL SECTION

■

Herein we described the discovery of EP2 antagonist 1 from a

HTS of our internal library. Lead compound 1 possessed a

70 nM binding aﬃnity for the hEP2 receptor and a 600 nM

IC₅₀value in the hEP2 cAMP assay. It was a potent inhibitor of

CYP3A4, had low microsomal stability, and lacked the exquisite

selectivity against the hEP3 and hEP4 receptors that we desired.

Subsequent modular SAR studies resulted in the discovery of

All solvents and chemicals used were reagent grade. Anhydrous

solvents were purchased from Aldrich and used as such. Analytical thin

layer chromatography (TLC) and ﬂash chromatography were

performed on Merck silica gel 60 (230−400 mesh). Removal of

solvents was conducted by using a rotary evaporator, and residual

solvent was removed from nonvolatile compounds using a vacuum

manifold maintained at approximately 1 Torr. All yields reported are

K

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Scheme 10

a

Reagents and conditions: (a) 60 or (R)-tert-butyl 1-hydroxypropan-2-ylcarbamate, DEAD, PPh₃, THF rt, 75−79%; (b) TFA, DCM, rt, quant; (c)

(i) benzo[b]thiophen-3-ylboronic acid, Pd(OAc)₂, SPhos, K₃PO₄, n-butanol, 100 °C, 76%, (ii) NaBH₄, MeOH, rt, quant (94), or (i) NaBH₄,

MeOH, rt, 72−92%, (ii) 5-ﬂuoro-1H-indole, (1S,2S)-(+)-N,N′-dimethyl-1,2-cyclohexanediamine, CuI, K₃PO₄, dioxane, 110 °C, 78%−quant (95 and

96); (d) 2-hydroxyisonicotinaldehyde, NaBH(OAc)₃, dichloroethane, rt, 33−92%.

isolated yields. Preparative reversed-phase−high pressure liquid

chromatography (RP−HPLC) was performed using an Agilent 1100

series HPLC and Phenomenex Gemini C18 column (5 μm, 100 mm ×

30 mm i.d.), eluting with a binary solvent system A and B using a

gradient elution [A, H₂O with 0.1% TFA; B, CH₃CN with 0.1% TFA]

with UV detection at 220 nm. All ﬁnal compounds were puriﬁed to

≥95% purity as determined by an Agilent 1100 series HPLC with UV

detection at 220 nm using the following method: Zorbax SB-C8

column (3.5 μm, 150 mm × 4.6 mm i.d.), eluting with a binary solvent

system A and B using a 5−95% B (0−15 min) gradient elution

[A, H₂O with 0.1% TFA; B, CH₃CN with 0.1% TFA]; ﬂow rate

1.5 mL/min. Mass spectral data was recorded on an Agilent 1100

series LCMS with UV detection at 254 nm. NMR spectra were

recorded on a Varian Gemini 400 MHz or Bruker Avance 500 MHz

NMR spectrometer. Chemical shifts (δ) are reported in parts per

million (ppm) relative to residual undeuterated solvent as internal

reference and coupling constants (J) are reported in hertz (Hz).

Splitting patterns are indicated as follows: s = singlet; d = doublet; t =

triplet; q = quartet; qn = quintet; dd = doublet of doublet; dt =

doublet of triplets; td = triplet of doublets; m = multiplet; br = broad

peak.

General Procedure for the Preparation of Benzoxazepines

1−16. Sodium triacetoxyborohydride (1.25 equiv) was added to a

0.1 M solution of benzoxazepine 59 (1 equiv) and the corresponding

aldehyde (1 equiv) in dichloroethane at rt. The reaction mixture was

concentrated in vacuo once the reaction was deemed complete by

TLC or LCMS. Puriﬁcation by ﬂash column chromatography provided

the desired products.

7-(Benzo[b]thiophen-3-yl)-9-methoxy-4-(pyridin-3-ylmeth-

yl)-2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (1). Following the

general reductive amination procedure using nicotinaldehyde provided

the title compound as a white solid in 61% yield. ¹H NMR (CDCl₃) δ

3.25 (m, 2H), 3.83 (m, 2H), 3.95 (s, 3H), 3.99 (m, 2H), 4.28 (m, 2H),

6.83 (d, J = 2.0 Hz, 1H), 7.12 (m, 1H), 7.34 (m, 1H), 7.40 (s, 1H),

7.44−7.41 (m, 2H), 7.95−7.89 (m, 3H), 8.58 (m, 2H). ESIMS m/z

(rel intensity) 403.0 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-9-methoxy-4-(pyridin-2-ylmeth-

yl)-2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (2). Following the

general reductive amination procedure using 2-pyridinecarboxaldehyde

1

provided the title compound as an oﬀ-white solid in 53% yield. H

NMR (CDCl₃) δ 3.22 (m, 2H), 3.89 (s, 2H), 3.93 (s, 3H), 3.97 (s,

2H), 4.25 (m, 2H), 6.85 (d, J = 2.0 Hz, 1H), 7.06 (d, J = 2.0 Hz, 1H),

7.19 (m, 1H), 7.36 (s, 1H), 7.42−7.39 (m, 2H), 7.47 (d, J = 7.8 Hz,

1H), 7.68 (dt, J = 1.8 and 7.7 Hz, 1H), 7.93−7.91 (m, 2H), 8.60 (m,

1H). ESIMS m/z (rel intensity) 403.0 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-9-methoxy-4-(pyridin-4-ylmeth-

yl)-2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (3). Following the

general reductive amination procedure using 4-pyridinecarboxaldehyde

provided the title compound as an oﬀ-white solid in 39% yield. ¹H NMR

(CDCl₃) δ 3.19 (m, 2H), 3.75 (s, 2H), 3.90 (s, 2H), 3.94 (s, 3H), 4.23

(m, 2H), 6.81 (d, J = 1.6 Hz, 1H), 7.08 (d, J = 1.6 Hz, 1H), 7.33 (d, J =

5.4 Hz, 2H), 7.36 (s, 1H), 7.41 (m, 2H), 7.93−7.88 (m, 2H), 8.57 (d, J =

5.6 Hz, 2H). ESIMS m/z (rel intensity) 403.0 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-9-methoxy-4-(pyrimidin-5-yl-

methyl)-2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (4). Follow-

ing the general reductive amination procedure using pyrimidine-5-

carboxaldehyde provided the title compound as a white solid in 24%

yield. ¹H NMR (CDCl₃) δ 3.22 (m, 2H), 3.77 (m, 2H), 3.95 (s, 5H),

4.25 (m, 2H), 6.83 (d, J = 2.0 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 7.39

(s, 1H), 7.43 (m, 2H), 7.95−7.89 (m, 2H), 8.76 (s, 2H), 9.17 (s, 1H).

ESIMS m/z (rel intensity) 404.0 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-9-methoxy-4-((2-methoxypyri-

din-3-yl)methyl)-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine (5).

Following the general reductive amination procedure using 2-

methoxynicotinaldehyde provided the title compound as a white

1

solid in 61% yield. H NMR (CDCl₃) δ 3.18 (m, 2H), 3.70 (s, 2H),

3.94 (br s, 8H), 4.25 (m, 2H), 6.89 (m, 2H), 7.08 (s, 1H), 7.37 (s,

1H), 7.40 (m, 2H), 7.73 (d, J = 6.7 Hz, 1H), 7.91 (m, 2H), 8.09 (m,

1H). ESIMS m/z (rel intensity) 433.1 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-9-methoxy-4-((6-methoxypyridin-3-

yl)methyl)-2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (6). Follow-

ing the general reductive amination procedure using 6-methoxynico-

tinaldehyde provided the title compound as a white solid in 75% yield.

L

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

¹H NMR (CDCl₃) δ 3.16 (m, 2H), 3.67 (s, 2H), 3.91 (s, 2H), 3.94 (s,

provided the title compound as a white solid in 70% yield. H NMR

(MeOD) δ 3.33 (m, 2H), 3.64 (m, 2H), 3.94 (s, 3H), 4.49 (br s, 2H),

4.57 (s, 2H), 7.13 (s, 1H), 7.34 (s, 1H), 7.42 (m, 2H), 7.50 (m, 3H),

7.61 (s, 1H), 7.64 (s, 1H), 7.92 (m, 1H), 7.95 (m, 1H). ESIMS m/z

(rel intensity) 436.1 ([M + H], 100).

3H), 3.96 (s, 3H), 4.23 (m, 2H), 6.76 (d, J = 8.6 Hz, 1H), 6.86 (s,

1H), 7.09 (s, 1H), 7.39 (s, 1H), 7.42 (m, 2H), 7.67 (m, 1H), 7.93 (m,

2H), 8.07 (s, 1H). ESIMS m/z (rel intensity) 433.1 ([M + H], 100).

5-((7-(Benzo[b]thiophen-3-yl)-9-methoxy-2,3-dihydrobenzo-

[f ][1,4]oxazepin-4(5H)-yl)methyl)pyridin-2-ol (7). Following the

general reductive amination procedure using 6-hydroxynicotinalde-

hyde provided the title compound as a white solid in 15% yield.

¹H NMR (CDCl₃) δ 3.14 (m, 2H), 3.48 (s, 2H), 3.87 (s, 2H), 3.92 (s,

3H), 4.19 (m, 2H), 6.60 (d, J = 9.3 Hz, 1H), 6.84 (s, 1H), 7.08 (s,

1H), 7.27 (s, 1H), 7.39 (m, 3H), 7.57 (d, J = 9.3 Hz, 1H), 7.90 (t,

J = 7.1 Hz, 2H), 12.94 (br s, 1H). ESIMS m/z (rel intensity) 312.1

([(M − CH₂C₅H₄NO) + H], 100), 419.0 ([M + H], 20).

3-((7-(Benzo[b]thiophen-3-yl)-9-methoxy-2,3-dihydrobenzo-

[f ][1,4]oxazepin-4(5H)-yl)methyl)benzonitrile (16). Following

the general reductive amination procedure using 3-formylbenzonitrile

1

provided the title compound as a white solid in 98% yield. H NMR

(CDCl₃) δ 3.17 (m, 2H), 3.76 (s, 2H), 3.89 (s, 2H), 3.94 (s, 3H), 4.23

(m, 2H), 6.79 (d, J = 1.5 Hz, 1H), 7.08 (d, J = 1.5 Hz, 1H), 7.37 (s,

1H), 7.50−7.39 (m, 3H), 7.58 (m, 2H), 7.72 (s, 1H), 7.90 (m, 2H).

ESIMS m/z (rel intensity) 427.1 ([M + H], 100).

General Procedure A for the Preparation of Benzoxazepines

17 and 23−26.³⁶A round-bottomed ﬂask was charged with a boronic

acid (1.0 equiv), aryl bromide 64 (1.0 equiv), K₃PO₄(3.0 equiv),

phosphine ligand, and palladium catalyst. The ﬂask was purged with

nitrogen, and n-butanol (1 mL/0.1 mol 64) was added. The reaction

mixture was heated at 100 °C for 12 h, cooled to rt, ﬁltered through

Celite, and washed with EtOAc. The ﬁltrate was concentrated in vacuo

and puriﬁed by ﬂash chromatography on silica gel or by reverse phase

HPLC.

General Procedure B for the Preparation of Benzoxazepines

18−22.³⁶A round-bottomed ﬂask was charged with a boronic acid

(1 equiv), aryl bromide 64 (1 equiv), Na₂CO₃(2.0 equiv), and SPhos

(0.1 equiv). The reaction ﬂask was purged with nitrogen followed by

the addition of Pd₂dba₃(0.1 equiv) and dioxane, water, and EtOH

(8:2:1, 0.5 mL/0.1 mol 64). The reaction ﬂask was purged with

nitrogen, and the reaction mixture was heated at 80 °C for 48 h. The

reaction mixture was concentrated in vacuo and puriﬁed by ﬂash

chromatography on silica gel or reverse-phase HPLC.

7-(Benzo[b]thiophen-3-yl)-4-benzyl-9-methoxy-2,3,4,5-

tetrahydrobenzo[f ][1,4]oxazepine (8). Following the general

reductive amination procedure using benzaldehyde provided the title

1

compound as a white solid in 64% yield. H NMR (CD₃OD) δ 3.30

(m, 2H), 3.65 (m, 2H), 3.93 (s, 3H), 4.52 (br s, 2H), 4.59 (br s, 2H),

7.13 (d, J = 1.7 Hz, 1H), 7.34 (d, J = 2.0 Hz, 1H), 7.42 (m, 2H), 7.52

(m, 3H), 7.57 (m, 2H), 7.61 (s, 1H), 7.90 (m, 1H), 7.96 (m, 1H).

ESIMS m/z (rel intensity) 402.1 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-4-(4-chlorobenzyl)-9-methoxy-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (9). Following the

general reductive amination procedure using 4-chlorobenzaldehyde

1

provided the title compound as a white solid in 49% yield. H NMR

(CDCl₃) δ 3.15 (m, 2H), 3.69 (s, 2H), 3.88 (s, 2H), 3.93 (s, 3H), 4.21

(m, 2H), 6.82 (d, J = 2.0 Hz, 1H), 7.07 (d, J = 2.0 Hz, 1H), 7.31 (br s,

4 H), 7.36 (s, 1H), 7.41 (m, 2H), 7.91 (m, 2H).

7-(Benzo[b]thiophen-3-yl)-9-methoxy-4-(4-methylbenzyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (10). Following the

general reductive amination procedure using 4-methylbenzaldehyde

1

provided the title compound as an oﬀ-white solid in 52% yield. H

General Procedure C for the Preparation of Benzoxazepines

27 and 30−31. Butyllithium (1.2 mL, 1.89 mmol) (1.5 M in

hexanes) was added dropwise to a solution of 64 (440 mg, 1.26 mmol)

in anhydrous THF (13 mL) at −78 °C, followed by the addition of

triisopropyl borate (436 μL, 1.89 mmol). The reaction mixture was

aged at −78 °C for 1 h, warmed to rt, and quenched with 1 M HCl

(1.5 mL). The reaction mixture was concentrated in vacuo and

puriﬁed by ﬂash chromatography on silica gel to provide (9-methoxy-

4-(pyridin-3-ylmethyl)-2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepin-7-yl)-

boronic acid, which was used directly in the next step. A reaction vessel

was charged with an arylhalide (1 equiv), (9-methoxy-4-(pyridin-3-

ylmethyl)-2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepin-7-yl)boronic acid

(1.0 equiv), K₃PO₄(3.0 equiv), and XPhos (0.1 equiv). The reaction

vessel was purged with nitrogen before adding n-butanol (1 mL/

0.1 mol 65) and Pd(OAc)₂(0.05 equiv). The reaction vessel was

purged with nitrogen and heated at 100 °C for 12 h. The reaction

mixture was concentrated in vacuo and puriﬁed by ﬂash chromatog-

raphy on silica gel or by reverse-phase HPLC.

General Procedure D for the Preparation of Benzoxazepines

29 and 32−38.⁴⁹An oven-dried reaction vessel was charged with

64 (1.0 equiv), an indole (1.5 equiv), K₃PO₄(2.2 equiv), and CuI

(0.1 equiv). The reaction vessel was purged with nitrogen before adding

(1S,2S)-(+)-N,N′-dimethyl-1,2-cyclohexanediamine (0.2 equiv) and

dioxane (1 mL/0.1 mol 64) and then purged again. The reaction

mixture was heated at 110 °C for 12 h, concentrated in vacuo, and

puriﬁed by ﬂash chromatography on silica gel.

NMR (DMSO-d₆) δ 2.33 (s, 3H), 3.48 (br s, 2H), 3.88 (s, 3H), 4.14

(m, 1H), 4.34 (m, 1H), 4.54 (m, 4H), 7.18 (s, 1H), 7.28 (d, J = 7.8

Hz, 2H), 7.32 (s, 1H), 7.46 (m, 2H), 7.51 (d, J = 7.5 Hz, 2H), 7.84 (s,

1H), 8.00 (m, 1H), 8.08 (m, 1H).

7-(Benzo[b]thiophen-3-yl)-9-methoxy-4-(4-methoxybenzyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (11). Following the

general reductive amination procedure using p-anisaldehyde provided

the title compound as a white solid in 95% yield. ¹H NMR (CDCl₃) δ

3.14 (m, 2H), 3.67 (s, 2H), 3.81 (s, 3H), 3.88 (s, 2H), 3.93 (s, 3H),

4.21 (m, 2H), 6.85 (m, 1H), 6.88 (d, J = 8.2 Hz, 2H), 7.06 (m, 1H),

7.28 (d, J = 8.2 Hz, 2H), 7.36 (s, 1H), 7.41 (m, 2H), 7.92 (m, 2H).

Methyl 4-((7-(Benzo[b]thiophen-3-yl)-9-methoxy-2,3-

dihydrobenzo[f ][1,4]oxazepin-4(5H)-yl)methyl)benzoate (12).

Following the general reductive amination procedure using methyl

4-formylbenzoate provided the title compound as a white solid in 25%

1

yield. H NMR (CDCl₃) δ 3.18 (m, 2H), 3.78 (s, 2H), 3.89 (s, 2H),

3.92 (s, 3H), 3.93 (s, 3H), 4.23 (m, 2H), 6.80 (d, J = 1.7 Hz, 1H), 7.07

(d, J = 1.7 Hz, 1H), 7.36 (s, 1H), 7.41 (m, 2H), 7.46 (d, J = 8.1 Hz,

2H), 7.91 (m, 2H), 8.02 (d, J = 8.3 Hz, 2H). ESIMS m/z (rel

intensity) 460.1 ([M + H], 100).

4-((7-(Benzo[b]thiophen-3-yl)-9-methoxy-2,3-dihydrobenzo-

[f ][1,4]oxazepin-4(5H)-yl)methyl)benzonitrile (13). Following

the general reductive amination procedure using methyl 4-

formylbenzonitrile provided the title compound as a white solid in

1

83% yield. H NMR (CDCl₃) δ 3.17 (m, 2H), 3.78 (s, 2H), 3.88 (s,

2H), 3.93 (s, 3H), 4.22 (m, 2H), 6.79 (s, 1H), 7.08 (s, 1H), 7.36 (s,

1H), 7.41 (m, 2H), 7.50 (d, J = 7.8 Hz, 2H), 7.63 (d, J = 7.8 Hz, 2H),

7.90 (m, 2H). ESIMS m/z (rel intensity) 427.1 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-9-methoxy-4-(4-(methylsulfonyl)-

benzyl)-2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (14). Follow-

ing the general reductive amination procedure using 4-(methylsulfonyl)-

benzaldehyde provided the title compound as a white solid in 55% yield.

¹H NMR (CDCl₃) δ 3.06 (s, 3H), 3.21 (m, 2H), 3.85 (s, 2H), 3.94 (s,

9-Methoxy-4-(pyridin-3-ylmethyl)-7-(thiophen-3-yl)-2,3,4,5-

tetrahydrobenzo[f ][1,4]oxazepine (17). Following general proce-

dure A using thiophene-3-boronic acid, XPhos (0.1 equiv), and

Pd(OAc)₂(0.05 equiv) provided the title compound as a white solid in

34% yield. ¹H NMR (CD₃CN) δ 3.59 (m, 2H), 3.91 (s, 3H), 4.28 (m,

2H), 4.45 (s, 2H), 4.53 (s, 2H), 7.13 (d, J = 2.4 Hz, 1H), 7.36 (d, J =

2.0 Hz, 1H), 7.44 (dd, J = 5.1 and 1.6 Hz, 1H), 7.50 (dd, J = 5.1 and

2.7 Hz, 1H), 7.61 (m, 1H), 7.98 (dd, J = 8.0 and 5.7 Hz, 1H), 8.53 (d,

J = 7.8 Hz, 1H), 8.83 (d, J = 5.1 Hz, 1H), 8.91 (s, 1H). ESIMS m/z

(rel intensity) 353.0 ([M + H], 100).

5H), 4.24 (m, 2H), 6.82 (d, J = 2.0 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H),

7.37 (s, 1H), 7.42 (m, 2H), 7.60 (d, J = 8.1 Hz, 2H), 7.88 (m, 1H), 7.92

(m, 3H). ESIMS m/z (rel intensity) 480.1 ([M + H], 100).

7-(Benzo[b]thiophen-2-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (18). Following general

procedure B using benzo[b]thiophen-2-ylboronic acid provided the

7-(Benzo[b]thiophen-3-yl)-4-(3-chlorobenzyl)-9-methoxy-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (15). Following the gen-

eral reductive amination procedure using 3-chlorobenzaldehyde

M

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

title compound as a white solid in 97% yield. H NMR (MeOD) δ

3.35 (m, 2H), 3.78 (m, 2H), 3.97 (s, 3H), 4.40 (m, 2H), 4.71 (s, 2H),

7.35 (m, 3H), 7.51 (s, 1H), 7.71 (s, 1H), 7.84 (m, 2H), 8.19 (s, 1H),

8.88 (s, 1H), 9.01 (s, 1H), 9.22 (s, 1H). ESIMS m/z (rel intensity)

403.1 ([M + H], 100).

(0.1 equiv) provided the title compound as a white solid in 68% yield.

¹H NMR (CD₃CN) δ 3.63 (m, 2H) 3.94 (s, 3H) 4.33 (m, 2H) 4.50 (s,

2H) 4.58 (s, 2H) 7.10 (d, J = 2.4 Hz, 1H) 7.34 (d, J = 2.4 Hz, 1H)

7.50 (d, J = 8.6 Hz, 2H) 7.62 (d, J = 8.6 Hz, 2H) 8.04 (br s, 1 H) 8.59

(d, J = 8.2 Hz, 1H) 8.89 (br s, 1H) 8.96 (br s, 1H). ESIMS m/z (rel

intensity) 381.1 ([M + H], 100).

9-Methoxy-7-phenyl-4-(pyridin-3-ylmethyl)-2,3,4,5-

tetrahydrobenzo[f ][1,4]oxazepine (19). Following general proce-

dure B using phenylboronic acid provided the title compound as a

white solid in 99% yield. ¹H NMR (CD₃CN) δ 3.62 (m, 2H), 3.92 (s,

3H), 4.31 (m, 2H), 4.51 (s, 2H), 4.59 (s, 2H), 7.11 (d, J = 2.0 Hz,

1H), 7.35 (d, J = 2.0 Hz, 1H), 7.39 (m, 1H), 7.47 (m, 2H), 7.63 (m,

2H), 8.04 (dd, J = 5.8 and 8.0 Hz, 1H), 8.61 (m, 1H), 8.85 (m, 1H),

8.95 (s, 1H).

7-(Benzo[d]isothiazol-3-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (27). Following general

procedure C using 3-chlorobenzo[d]isothiazole provided the title

1

compound as a white solid in 24% yield. H NMR (CD₃CN) δ 3.12

(m, 2H), 3.74 (s, 2H), 3.91 (s, 2H), 3.92 (s, 3H), 4.14 (m, 2H), 7.14

(d, J = 2.2 Hz, 1H), 7.33 (ddd, J = 7.6, 4.7, and 0.6 Hz, 1H), 7.44 (d,

J = 2.2 Hz, 1H), 7.55 (ddd, J = 1.0, 7.0, and 8.2 Hz, 1H), 7.64 (ddd, J =

1.0, 7.0, and 8.2 Hz, 1H), 7.74 (dt, J = 7.8 and 1.9 Hz, 1H), 8.13 (dt,

J = 8.2 and 0.9 Hz, 1H), 8.23 (dt, J = 8.2 and 1.0 Hz, 1H), 8.53 (d, J =

1.8 Hz, 1H). ESIMS m/z (rel intensity) 404.2 ([M + H], 100).

7-(1H-Benzo[d]imidazol-1-yl)-9-methoxy-4-(pyridin-3-yl-

methyl)-2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (28). To a

glass vial containing CuI (2.7 mg, 0.014 mmol), benzimidazole

(19 mg, 0.165 mmol), 64 (50 mg, 0.143 mmol), 8-hydroxyquinoline

(2.1 mg, 0.014 mmol), and Cs₂CO₃(47 mg, 0.143 mmol) was added

DMF (1 mL) and water (0.1 mL).⁵⁰The vial was purged with nitrogen,

capped, and heated at 130 °C for 24 h. The reaction mixture was

diluted with 1 M LiCl (2 mL) and extracted with EtOAc (3 × 2 mL).

The organics were pooled, dried over Na₂SO₄, ﬁltered, and con-

centrated in vacuo. Puriﬁcation by reverse phase HPLC followed by

neutralization of the product and ﬁltration through a plug of silica gel

9-Methoxy-7-(naphthalen-1-yl)-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (20). Following gen-

eral procedure B using naphthalen-1-ylboronic acid provided the TFA

1

salt of the title compound as a white solid in 81% yield. H NMR

(D₂O) δ 3.67 (s, 3H), 3.74 (m, 2H), 4.33 (s, 2H), 4.34 (m, 2H), 4.63

(s, 2H), 6.69 (d, J = 1.6 Hz, 1H), 7.00 (d, J = 1.6 Hz, 1H), 7.25 (d, J =

6.3 Hz, 1H), 7.35 (m, 1 H), 7.46 (m, 2H), 7.66 (d, J = 8.6 Hz, 1H),

7.86 (d, J = 8.2 Hz, 1H), 7.90 (d, J = 8.2 Hz, 1H), 8.08 (dd, J = 5.9 and

8.2 Hz, 1H), 8.63 (dt, J = 1.6 and 8.6 Hz, 1H), 8.87 (d, J = 5.1 Hz,

1H), 8.92 (d, J = 1.6 Hz, 1H). ESIMS m/z (rel intensity) 397.1 ([M + H],

100).

9-Methoxy-4-(pyridin-3-ylmethyl)-7-(o-tolyl)-2,3,4,5-

tetrahydrobenzo[f ][1,4]oxazepine (21). Following general proce-

dure B using o-tolylboronic acid provided the title compound as a

white solid in 78% yield. ¹H NMR (CD₃CN) δ 2.26 (s, 3H), 3.08 (m,

2H), 3.67 (s, 2H), 3.80 (m, 2H), 3.82 (s, 3H), 4.06 (m, 2H), 6.53 (d,

J = 2.0 Hz, 1H), 6.87 (d, J = 2.0 Hz, 1H), 7.30−7.19 (m, 5H), 7.69 (m,

1H), 8.45 (m, 2H). ESIMS m/z (rel intensity) 361.1 ([M + H], 100).

9-Methoxy-4-(pyridin-3-ylmethyl)-7-(m-tolyl)-2,3,4,5-

tetrahydrobenzo[f ][1,4]oxazepine (22). Following general proce-

dure B using m-tolylboronic acid provided the TFA salt of the title

1

provided the title compound as a white solid in 35% yield. H NMR

(CD₃CN) δ 3.13 (m, 2H), 3.74 (s, 2H), 3.89 (s, 2H), 3.90 (s, 3H),

4.12 (m, 2H), 6.87 (d, J = 2.5 Hz, 1H), 7.16 (d, J = 2.5 Hz, 1H), 7.35

(m, 3H), 7.60 (m, 1H), 7.76 (m, 2H), 8.19 (s, 1H), 8.48 (dd, J = 4.7

and 1.4 Hz, 1H), 8.52 (d, J = 1.4 Hz, 1H). ESIMS m/z (rel intensity)

387.2 ([M + H], 100).

7-(1H-Indol-1-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-2,3,4,5-

tetrahydrobenzo[f ][1,4]oxazepine (29). Following general proce-

dure D using indole provided the title compound as a white solid in

1

compound as a white solid in 89% yield. H NMR (D₂O) δ 2.35 (s,

3H), 3.78 (m, 2H), 3.87 (s, 3H), 4.36 (m, 2H), 4.54 (s, 2H), 4.77 (s,

2H), 7.03 (s, 1H), 7.24 (m, 1H), 7.26 (s, 1H), 7.37 (m, 2H), 8.23 (m,

1H), 8.80 (d, J = 7.6 Hz, 1H), 8.97 (d, J = 5.9 Hz, 1H), 9.03 (s, 1H).

ESIMS m/z (rel intensity) 361.1 ([M + H], 100).

9-Methoxy-4-(pyridin-3-ylmethyl)-7-(p-tolyl)-2,3,4,5-

tetrahydrobenzo[f ][1,4]oxazepine (23). Following general proce-

dure A using 4-methylphenylboronic acid, XPhos (0.1 equiv), and

Pd(OAc)₂(0.05 equiv) provided the title compound as a white solid in

1

33% yield. H NMR (CD₃CN) δ 3.12 (m, 2H), 3.73 (s, 2H), 3.88 (s,

2H), 3.89 (s, 3H), 4.11 (m, 2H), 6.69 (dd, J = 3.3 and 0.8 Hz, 1H),

6.80 (d, J = 2.4 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 7.16 (m, 1H), 7.24

(m, 1H), 7.33 (m, 1H), 7.45 (d, J = 3.3 Hz, 1H), 7.57 (m, 1H), 7.67

(dt, J = 8.0 and 0.9 Hz, 1H), 7.74 (dt, J = 7.7 and 2.0 Hz, 1H), 8.49

(dd, J = 4.7 and 1.6 Hz, 1H), 8.5 (d, J = 1.6 Hz, 1H). ESIMS m/z (rel

intensity) 386.2 ([M + H], 100).

1

37% yield. H NMR (CD₃CN) δ 2.39 (s, 3H), 3.09 (m, 2H), 3.70 (s,

7-(1H-Indol-3-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-2,3,4,5-

tetrahydrobenzo[f ][1,4]oxazepine (30). Following general proce-

dure C using tert-butyl 3-bromo-1H-indole-1-carboxylate provided tert-

butyl 3-(9-methoxy-4-(pyridin-3-ylmethyl)-2,3,4,5-tetrahydrobenzo-

[f ][1,4]oxazepin-7-yl)-1H-indole-1-carboxylate as a white solid in

45% yield. A solution of the Boc-protected indole and TFA (0.14 mL,

1.8 mmol) in DCM (1 mL) was stirred at rt for 12 h to provide the

2H), 3.86 (s, 2H), 3.90 (s, 3H), 4.07 (m, 2H), 6.87 (d, J = 2.2 Hz,

1H), 7.16 (d, J = 2.2 Hz, 1H), 7.28 (m, 2H), 7.34 (m, 1H), 7.51 (d, J =

8.0 Hz, 2H), 7.73 (dt, J = 7.8 and 2.0 Hz, 1H), 8.50 (m, 2H). ESIMS

m/z (rel intensity) 361.1 ([M + H], 100).

7-(2-Chlorophenyl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (24). Following gen-

eral procedure A using 2-chlorophenylboronic acid and Pd(dppf)Cl₂

(0.1 equiv) provided the title compound as a white solid in 95% yield.

¹H NMR (CDCl₃) δ 3.67 (m, 2H), 3.89 (s, 3H), 4.35 (m, 2H), 4.55

(s, 2H), 4.66 (s, 2H), 6.95 (d, J = 2.0 Hz, 1H), 7.24 (d, J = 2.0 Hz,

1H), 7.42 (m, 3H), 7.55 (m, 1H), 8.11 (dd, J = 7.8 and 5.9 Hz, 1H),

8.68 (d, J = 8.2 Hz, 1H), 8.87 (d, J = 5.5 Hz, 1H), 8.98 (s, 1H). ESIMS

m/z (rel intensity) 381.1 ([M + H], 100).

1

title compound as a white solid in 24% yield. H NMR (CD₃CN) δ

3.00 (m, 2H), 3.62 (s, 2H), 3.77 (s, 2H), 3.81 (s, 3H), 3.97 (m, 2H),

6.83 (d, J = 2.0 Hz, 1H), 7.05 (m, 1H), 7.12 (m, 2H), 7.24 (ddd, J =

7.6, 4.7, and 0.6 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 7.40 (dt, J = 8.2 and

0.9 Hz, 1H), 7.64 (dt, J = 7.8 and 2.0 Hz, 1H), 7.77 (d, J = 8.4 Hz,

1H), 8.40 (dd, J = 4.7 and 1.8 Hz, 1H), 8.42 (d, J = 1.6 Hz, 1H), 9.36

(s, 1H). ESIMS m/z (rel intensity) 386.2 ([M + H], 100).

7-(3-Chlorophenyl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (25). Following gen-

eral procedure A using 3-chlorophenylboronic acid and Pd(dppf)Cl₂

(0.1 equiv) provided the title compound as a white solid in 64% yield.

¹H NMR (CD₃CN) δ 3.63 (m, 2H), 3.95 (s, 3H), 4.33 (m, 2H), 4.51

(s, 2H), 4.60 (s, 2H), 7.13 (d, J = 2.4 Hz, 1H), 7.36 (d, J = 2.4 Hz,

1H), 7.42 (m, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.58 (dt, J = 7.4 and 1.2

Hz, 1H), 7.68 (t, J = 1.8 Hz, 1H), 8.05 (m, 1H), 8.61 (d, J = 8.2 Hz,

1H), 8.88 (br s, 1H), 8.97 (br s, 1H). ESIMS m/z (rel intensity) 381.1

([M + H], 100).

9-Methoxy-7-(1-methyl-1H-indol-3-yl)-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (31). Following general

procedure C using 3-bromo-1H-indole provided the title compound as

1

a white solid in 31% yield. H NMR (CD₃CN) δ 3.35 (m, 2H), 3.86

(s, 3H), 3.93 (s, 3H), 4.10 (s, 2H), 4.19 (s, 2H), 4.20 (m, 2H), 7.04

(d, J = 2.0 Hz, 1H), 7.18 (m, 1H), 7.29 (m, 2H), 7.48−7.44 (m, 3H),

7.92 (m, 1H), 7.95 (dt, J = 8.0 and 1.9 Hz, 1H), 8.63 (m, 2H). ESIMS

m/z (rel intensity) 400.3 ([M + H], 100).

7-(3-Chloro-1H-indol-1-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (32). Following general

procedure D using 3-chloro-1H-indole provided the title compound as

7-(4-Chlorophenyl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (26). Following gen-

eral procedure A using 4-chlorophenylboronic acid and Pd(dppf)Cl₂

1

a white solid in 26% yield. H NMR (CD₃CN) δ 3.12 (m, 2H), 3.73

(s, 2H), 3.87 (s, 2H), 3.88 (s, 3H), 4.11 (m, 2H), 6.79 (d, J = 2.5 Hz,

N

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1H), 7.09 (d, J = 2.5 Hz, 1H), 7.27 (m, 1H), 7.33 (m, 2H), 7.53 (s,

1H), 7.58 (m, 1H), 7.66 (m, 1H), 7.74 (dt, J = 7.8 and 2.0 Hz, 1H),

8.49 (dd, J = 4.7 and 1.6 Hz, 1H), 8.52 (d, J = 1.8 Hz, 1H). ESIMS

m/z (rel intensity) 420.1 ([M + H], 100).

(m, 2H), 3.09 (t, J = 8.5 Hz, 2H), 3.68 (s, 2H), 3.78 (s, 2H), 3.84 (s,

3H), 3.91 (t, J = 8.5 Hz, 2H), 4.01 (m, 2H), 6.46 (d, J = 2.7 Hz, 1H),

6.78 (d, J = 2.5 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 7.02 (dd, J = 8.4 and

2.2 Hz, 1H), 7.14 (m, 1H), 7.33 (dd, J = 7.7 and 4.8 Hz, 1H), 7.71 (dt,

J = 7.8 and 1.9 Hz, 1H), 8.50 (m, 2H). ESIMS m/z (rel intensity)

422.2 ([M + H], 100).

7-(4-Chloro-1H-indol-1-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (33). Following general

procedure D using 4-chloro-1H-indole provided the title compound as

7-(Benzo[b]thiophen-3-yl)-4-(pyridin-3-ylmethyl)-2,3,4,5-

tetrahydrobenzo[f ][1,4]oxazepine (40). NaBH(OAc)₃(339 mg,

1.60 mmol) was added to a solution of 71 (300 mg, 1.07 mmol) and

nicotinaldehyde (101 μL, 1.07 mmol) in dichloroethane (20 mL) at rt.

The reaction mixture was aged at rt for 12 h before adding DCM

(75 mL) and washing with saturated aqueous NaHCO₃and brine. The

solution was dried over MgSO₄, ﬁltered, and concentrated in vacuo to

provide an orange oil. Puriﬁcation by ﬂash chromatography on silica

gel eluting with 2% MeOH/DCM provided a colorless oil that was

dissolved in ether, and 1 M HCl in ether (1.4 mL) was added to

provide the title compound as a white solid (218 mg, 50%). ¹H NMR

(D₂O) δ 3.72 (m, 2H), 4.38 (m, 2H), 4.50 (s, 2H), 4.64 (s, 2H), 7.24

(d, J = 8.2 Hz, 1H), 7.38 (d, J = 2.0 Hz, 1H), 7.42 (m, 2H), 7.53 (s,

1H), 7.60 (dd, J = 8.2 and 2.0 Hz, 1H), 7.81 (m, 1H), 7.97 (m, 2H),

8.48 (d, J = 7.8 Hz, 1H), 8.80 (d, J = 4.7 Hz, 1H), 8.83 (s, 1H). ESIMS

m/z (rel intensity) 373.0 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-4-(pyridin-3-ylmethyl)-2,3,4,5-

tetrahydrobenzo[f ][1,4]oxazepin-9-ol (41). Following general

procedure A using 73 and benzo[b]thiophen-3-ylboronic acid,

XPhos (0.1 equiv), and Pd(OAc)₂(0.05 equiv) provided the title

compound as a white solid in 69% yield. ¹H NMR (CDCl₃) δ 3.19 (m,

2H), 3.75 (s, 2H), 3.90 (s, 2H), 4.18 (m, 2H), 6.74 (d, J = 2.2 Hz,

1H), 7.15 (d, J = 2.0 Hz, 1H), 7.31 (dd, J = 7.8 and 4.9 Hz, 1H), 7.34

(s, 1H), 7.38 (m, 2H), 7.76 (br d, J = 7.8 Hz, 1H), 7.90 (m, 2H), 8.55

(d, J = 4.2 Hz, 1H), 8.58 (s, 1H). ESIMS m/z (rel intensity) 389.0

([M + H], 100).

1

a white solid in 58% yield. H NMR (CD₃CN) δ 3.13 (m, 2H), 3.75

(s, 2H), 3.88 (s, 3H), 3.89 (s, 2H), 4.12 (m, 2H), 6.76 (dd, J = 3.1 and

0.8 Hz, 1H), 6.79 (d, J = 2.8 Hz, 1H), 7.10 (d, J = 2.4 Hz, 1H), 7.19 (s,

1H), 7.20 (d, J = 2.0 Hz, 1H), 7.34 (dd, J = 7.6 and 4.9 Hz, 1H), 7.49

(m, 1H), 7.51 (d, J = 3.5 Hz, 1H), 7.76 (m, 1H), 8.50 (br s, 1H), 8.53

(br s, 1H). ESIMS m/z (rel intensity) 420.1 ([M + H], 100).

7-(5-Chloro-1H-indol-1-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (34). Following general

procedure D using 5-chloro-1H-indole provided the title compound as

1

a white solid in 65% yield. H NMR (CD₃CN) δ 3.15 (m, 2H), 3.79

(s, 2H), 3.88 (s, 3H), 3.91 (s, 2H), 4.12 (m, 2H), 6.64 (dd, J = 3.1 and

0.8 Hz, 1H), 6.77 (d, J = 2.4 Hz, 1H), 7.08 (d, J = 2.8 Hz, 1H), 7.19

(dd, J = 8.8 and 2.2 Hz, 1H), 7.37 (m, 1H), 7.48 (d, J = 3.1 Hz, 1H),

7.51 (d, J = 8.6 Hz, 1H), 7.66 (d, J = 2.4 Hz, 1H), 7.78 (d, J = 7.8 Hz,

1H), 8.55 (m, 2H). ESIMS m/z (rel intensity) 420.1 ([M + H], 100).

7-(6-Chloro-1H-indol-1-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (35). Following general

procedure D using 6-chloro-1H-indole provided the title compound as

1

a white solid in 68% yield. H NMR (CD₃CN) δ 3.17 (m, 2H), 3.81

(s, 2H), 3.88 (s, 3H), 3.93 (s, 2H), 4.14 (m, 2H), 6.68 (dd, J = 3.3 and

0.8 Hz, 1H), 6.80 (d, J = 2.4 Hz, 1H), 7.08 (d, J = 2.4 Hz, 1H), 7.14

(dd, J = 8.4 and 2.0 Hz, 1H), 7.40 (br s, 1H), 7.44 (d, J = 3.1 Hz, 1H),

7.55 (m, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H), 8.5

(br s, 2H). ESIMS m/z (rel intensity) 420.1 ([M + H], 100).

7-(7-Chloro-1H-indol-1-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (36). Following general

procedure D using 7-chloro-1H-indole provided the title compound as

7-(Benzo[b]thiophen-3-yl)-9-ethoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (42). NaH (60% in

mineral oil) (4.2 mg, 0.106 mmol) was added to a solution of 41

(41 mg, 0.106 mmol) in anhydrous DMF (1 mL) at rt followed by the

addition of bromoethane (8 μL, 0.106 mmol). The reaction mixture

was stirred at rt for 12 h and diluted with water (2 mL), DCM (2 mL),

and brine (1 mL). The layers were separated, and the aqueous layer

was extracted with EtOAc. The organics were combined, dried over

Na₂SO₄, ﬁltered, and concentrated in vacuo. Puriﬁcation by ﬂash

chromatography on silica gel eluting with 0−10% MeOH/DCM

1

a white solid in 36% yield. H NMR (CD₃CN) δ 3.13 (m, 2H), 3.70

(s, 2H), 3.83 (s, 3H), 3.85 (s, 2H), 4.11 (m, 2H), 6.69 (m, 2H), 7.04

(d, J = 2.4 Hz, 1H), 7.10 (dd, J = 7.8 and 7.6 Hz, 1H), 7.21 (dd, J = 7.6

and 1.2 Hz, 1H), 7.29 (m, 2H), 7.63 (dd, J = 7.9 and 1.1 Hz, 1H), 7.71

(dt, J = 7.8 and 1.9 Hz, 1H), 8.46 (br s, 1H), 8.50 (br s, 1H). ESIMS

m/z (rel intensity) 420.1 ([M + H], 100).

1 - ( 9 - M e t h o x y - 4 - ( p y r i d i n - 3 - y l m e t h y l ) - 2 , 3 , 4 , 5 -

tetrahydrobenzo[f ][1,4]oxazepin-7-yl)-1H-indole-5-carbonitrile

(37). Following general procedure D using 1H-indole-5-carbonitrile

1

provided the title compound as a white solid (19.4 mg, 44%). H

provided the title compound as a white solid in 88% yield. H NMR

NMR (CD₃CN) δ 1.39 (t, J = 6.9 Hz, 3H), 3.10 (m, 2H), 3.71 (s,

2H), 3.84 (s, 2H), 4.09 (m, 2H), 4.12 (q, J = 6.9 Hz, 2H), 6.83 (d, J =

2.2 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.31 (m, 1H), 7.43 (m, 2H),

7.51 (s, 1H), 7.72 (dt, J = 7.8 and 2.0 Hz, 1H), 7.93 (m, 1H), 7.96 (m,

1H), 8.46 (dd, J = 4.7 and 1.6 Hz, 1H), 8.50 (d, J = 1.7 Hz, 1H).

ESIMS m/z (rel intensity) 417.3 ([M + H], 100).

(CD₃CN) δ 3.43 (m, 2H), 3.90 (s, 3H), 4.24 (s, 2H), 4.26 (s, 2H),

4.27 (m, 2H), 6.82 (dd, J = 3.3 and 0.8 Hz, 1H), 6.95 (d, J = 2.4 Hz,

1H), 7.19 (d, J = 2.4 Hz, 1H), 7.51 (dd, J = 8.7 and 1.7 Hz, 1H), 7.61

(d, J = 3.3 Hz, 1H), 7.64 (br s, 1H), 7.68 (d, J = 8.6 Hz, 1H), 8.11 (d,

J = 1.0 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.72 (br s, 2H). ESIMS m/z

(rel intensity) 411.2 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-9-isopropoxy-4-(pyridin-3-yl-

methyl)-2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (43). NaH

(60% in mineral oil) (6.6 mg, 0.154 mmol) was added to a solution

of 41 (60 mg, 0.154 mmol) in anhydrous DMF (1 mL) at rt followed

by the addition of 2-bromopropane (15 μL, 0.154 mmol). The

reaction mixture was stirred at rt for 12 h and diluted with water

(2 mL), DCM (2 mL), and brine (1 mL). The layers were separated,

and the aqueous layer was extracted with EtOAc. The organics were

combined, dried over Na₂SO₄, ﬁltered, and concentrated in vacuo.

Puriﬁcation by ﬂash chromatography on silica gel eluting with 0−10%

MeOH/DCM provided the title compound as a white solid (6.0 mg,

7-(5-Fluoro-1H-indol-1-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (38). Following general

procedure D using 5-ﬂuoro-1H-indole provided the title compound as

1

a white solid in 82% yield. H NMR (CD₃CN) δ 3.23 (m, 2H), 3.89

(s, 3H), 3.91 (s, 2H), 4.01 (s, 2H), 4.16 (m, 2H), 6.67 (d, J = 3.1 Hz,

1H), 6.83 (d, J = 2.5 Hz, 1H), 7.01 (td, J = 9.2 and 2.6 Hz, 1H), 7.12

(d, J = 2.5 Hz, 1H), 7.36 (dd, J = 9.8 and 2.5 Hz, 1H), 7.42 (dd, J = 7.7

and 5.0 Hz, 1H), 7.50 (d, J = 3.3 Hz, 1H), 7.54 (dd, J = 9.1 and 4.4 Hz,

1H), 7.87 (dt, J = 7.8 and 1.9 Hz, 1H), 8.56 (d, J = 3.9 Hz, 1H), 8.59

(s, 1H). ESIMS m/z (rel intensity) 404.2 ([M + H], 100).

7-(5-Chloroindolin-1-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (39). An oven-dried

glass vial was charged with 64 (83 mg, 0.238 mmol), sodium tert-

butoxide (69 mg, 0.713 mmol), 1,3-bis(2,6-diisopropylphenyl)-

imidazolidine hydrochloride (10 mg, 0.024 mmol), and Pd(OAc)₂

(2.7 mg, 0.012 mmol). The vial was purged with nitrogen, dioxane

(1.5 mL) and 5-chloroindoline (37 μL, 0.238 mmol) were added, the

vial was purged with nitrogen again, and then capped and heated at

110 °C for 12 h. The reaction mixture was concentrated in vacuo and

puriﬁed by ﬂash chromatography on silica gel to provide the title

1

9%). H NMR (CD₃CN) δ 1.33 (d, J = 6.1 Hz, 6H), 3.09 (m, 2H),

3.71 (s, 2H), 3.84 (s, 2H), 4.08 (m, 2H), 4.59 (sep, J = 6.1 Hz, 1H),

6.86 (d, J = 2.2 Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H), 7.31 (m, 1H), 7.43

(m 2H), 7.51 (s, 1H), 7.72 (dt, J = 7.8 and 2.0 Hz, 1H), 7.90 (m, 1H),

7.97 (m, 1H), 8.47 (dd, J = 4.8 and 1.7 Hz, 1H), 8.50 (d, J = 1.6 Hz,

1H). ESIMS m/z (rel intensity) 431.2 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-9-methyl-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (44). NaBH(OAc)₃

(323 mg, 1.52 mmol) was added to a solution of 72 (300 mg,

1.02 mmol) and nicotinaldehyde (96 μL, 1.02 mmol) in dichloroethane

1

compound as a white solid in 20% yield. H NMR (CD₃CN) δ 3.08

O

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(20 mL) at rt. The reaction mixture was aged at rt for 12 h before

adding DCM (75 mL) and washing with saturated aqueous NaHCO₃

and brine. The solution was dried over MgSO₄, ﬁltered, and con-

centrated in vacuo to provide an orange oil. Puriﬁcation by ﬂash

chromatography on silica gel eluting with 2% MeOH/DCM provided a

colorless oil that was dissolved in ether, and 1 M HCl in ether (1.5 mL)

was added to provide the title compound as a white solid (288 mg,

67%). ¹H NMR (D₂O) δ 2.17 (s, 3H), 3.67 (m, 2H), 4.31 (br s, 2H),

4.37 (s, 2H), 4.61 (s, 2H), 7.09 (br s, 1H), 7.35 (m, 4H), 7.71 (dd, J =

6.3 and 2.8 Hz, 1H), 7.90 (dd, J = 6.3 and 2.4 Hz, 1H), 8.06 (dd, J = 8.0

and 5.7 Hz, 1H), 8.61 (br d, J = 8.2 Hz, 1H), 8.83 (d, J = 5.6 Hz, 1H),

8.89 (s, 1H). ESIMS m/z (rel intensity) 387.1 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-9-chloro-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepine (45). Following the

general reductive amination procedure using 79 and nicotinaldehyde

mineral oil) (4.5 mg, 0.117 mmol) was added to a solution of 90

(19 mg, 0.058 mmol) in anhydrous THF (1 mL) at rt followed by

the addition of 3-(bromomethyl)pyridine hydrobromide (15 mg,

0.058 mmol). The reaction mixture was stirred at rt for 12 h, quenched

with water (1 mL), and extracted with EtOAc (3 × 2 mL). The

organics were combined, dried over Na₂SO₄, ﬁltered, and concentrated

in vacuo. Puriﬁcation by ﬂash chromatography on silica gel eluting

with 0−10% MeOH/DCM provided the title compound as a white

1

solid (6.9 mg, 28%). H NMR (CDCl₃) δ 3.56 (t, J = 5.5 Hz, 2H),

3.89 (s, 3H), 4.26 (t, J = 5.5 Hz, 2H), 4.82 (s, 2H), 7.34 (dd, J = 7.9

and 4.9 Hz, 1H), 7.36 (d, J = 2.4 Hz, 1H), 7.48−7.42 (m, 3H), 7.63 (s,

1H), 7.76 (m, 1H), 7.99 (m, 2H), 8.51 (dd, J = 4.6 and 1.5 Hz, 1H),

8.60 (d, J = 1.8 Hz, 1H). ESIMS m/z (rel intensity) 417.1 ([M + H], 100).

4-((7-(Benzo[b]thiophen-3-yl)-9-chloro-2,3-dihydrobenzo[f ]-

[1,4]oxazepin-4(5H)-yl)methyl)pyridin-2(1H)-one (51). Follow-

ing the general reductive amination procedure using 79 and

2-hydroxyisonicotinaldehyde provided the title compound as a white

solid in 92% yield. ¹H NMR (DMSO-d₆) δ 2.58 (br s, 2H), 3.77 (br s,

4H), 4.33 (br s, 2H), 6.26 (d, J = 6.7 Hz, 1H), 6.41 (br s, 1H), 7.53−

7.43 (m, 4H), 7.72 (br s, 1H), 7.89 (s, 1H), 7.91 (m, 1H), 8.09 (d, J =

7.4 Hz, 1H). ESIMS m/z (rel intensity) 423.1 ([M + H], 100).

4-((9-Chloro-7-(5-ﬂuoro-1H-indol-1-yl)-2,3-dihydrobenzo[f ]-

[1,4]oxazepin-4(5H)-yl)methyl)pyridin-2(1H)-one (52). Follow-

ing the general reductive amination procedure, 95 (13.15 g, 41.5 mmol)

and 2-hydroxyisonicotinaldehyde (5.62 g, 45.7 mmol) provided the

title compound as an oﬀ-white solid (10.0 g, 57%). ¹H NMR (CDCl₃)

δ 3.20 (m, 2H), 3.60 (s, 2H), 3.90 (s, 2H), 4.23 (m, 2H), 6.43 (dd,

J = 6.7 and 1.2 Hz, 1H), 6.56 (br s, 1H), 6.62 (dd, J = 3.5 and

0.8 Hz, 1H), 7.01 (td, J = 9.1 and 2.5 Hz, 1H), 7.07 (d, J = 2.8 Hz,

1H), 7.29 (m, 2H), 7.34 (d, J = 6.7 Hz, 1H), 7.40 (dd, J = 9.0 and 4.3

Hz, 1H), 7.45 (d, J = 2.7 Hz, 1H). ESIMS m/z (rel intensity) 424.2

([M + H], 100).

1

provided the title compound as a white solid in 45% yield. H NMR

(CDCl₃) δ 3.20 (m, 2H), 3.74 (s, 2H), 3.91 (s, 2H), 4.24 (m, 2H),

7.10 (d, J = 2.2 Hz, 1H), 7.29 (dd, J = 7.7 and 4.8 Hz, 1H), 7.37 (s,

1H), 7.42 (m, 2H), 7.55 (d, J = 2.0 Hz, 1H), 7.74 (br d, J = 8.1 Hz, 1H),

7.85 (m, 2H), 7.91 (m, 1H), 8.54 (dd, J = 4.7 and 1.3 Hz, 1H), 8.56 (d,

J = 1.7 Hz, 1H). ESIMS m/z (rel intensity) 407.1 ([M + H], 100).

8-(Benzo[b]thiophen-3-yl)-2-(pyridin-3-ylmethyl)-2,3,4,5-

tetrahydro-1H-benzo[c]azepine (46). Following the general

reductive amination procedure using 83 and nicotinaldehyde provided

the title compound as a white solid in 44% yield. ¹H NMR (CDCl₃) δ

1.79 (m, 2H), 3.00 (m, 2H), 3.12 (m, 2H), 3.61 (s, 2H), 3.91 (m, 2H),

7.14 (d, J = 1.8 Hz, 1H), 7.29 (m, 2H), 7.42 (m, 3H), 7.50 (s, 1H),

7.69 (dt, J = 7.8 and 2.0 Hz, 1H), 7.86 (m, 1H), 7.98 (m, 1H), 8.45

(m, 2H). ESIMS m/z (rel intensity) 371.2 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-4-(pyridin-3-ylmethyl)-2,3,4,5-

tetrahydro-1H-benzo[e][1,4]diazepine (47). Following the gen-

eral reductive amination procedure using 85 and nicotinaldehyde

1

(R)-4-((7-(Benzo[b]thiophen-3-yl)-9-chloro-3-methyl-2,3-

dihydrobenzo[f ][1,4]oxazepin-4(5H)-yl)methyl)pyridin-2(1H)-

one (53). Following the general reductive amination procedure, 94

(100 mg, 0.303 mmol) and 2-hydroxyisonicotinaldehyde (37.3 mg,

0.303 mmol) provided the title compound as a white solid (43.2 mg,

provided the title compound as a white solid in 53% yield. H NMR

(CD₃CN) δ 3.45 (s, 4H), 4.46 (s, 2H), 4.58 (s, 2H), 7.06 (d, J = 8.2

Hz, 1H), 7.44 (m, 1H), 7.47 (m, 2H), 7.52 (s, 1H), 7.55 (dd, J = 8.2

and 2.0 Hz, 1H), 7.93 (m, 1H), 7.95 (m, 1H), 8.00 (m, 1H), 8.49 (d,

J = 7.8 Hz, 1H), 8.84 (br s, 1H), 8.94 (br s, 1H). ESIMS m/z (rel

intensity) 372.1 ([M + H], 100).

1

33%). H NMR (CDCl₃) δ 1.22 (d, J = 7.0 Hz, 3H), 3.20 (m, 1H),

3.41 (d, J = 15.7 Hz, 1H), 3.54 (d, J = 15.7 Hz, 1H), 3.67 (d, J = 15.7

Hz, 1H), 3.96 (dd, J = 12.9 and 6.3 Hz, 1H), 4.16 (m, 2H), 6.30 (d, J =

6.7 Hz, 1H), 6.47 (s, 1H), 6.99 (d, J = 2.0 Hz, 1H), 7.23 (d, J = 6.7 Hz,

1H), 7.34−7.28 (m, 3H), 7.44 (d, J = 2.4 Hz, 1H), 7.73 (d, J =

7.4 Hz, 1H), 7.79 (d, J = 7.4 Hz, 1H). ESIMS m/z (rel intensity) 437.0

([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-1-methyl-4-(pyridin-3-ylmethyl)-

2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine (48). To a mixture

of 47 (54 mg, 0.111 mmol), formaldehyde (0.111 mmol), and 3 Å

molecular sieves in MeOH at rt was added NaCNBH₃(10 mg,

0.167 mmol). The reaction mixture was stirred at rt for 48 h, con-

centrated in vacuo, and puriﬁed by ﬂash chromatography on silica gel

eluting with 0−10% MeOH/DCM to provide the title compound as a

white solid (14.2 mg, 33%). ¹H NMR (CD₃CN) δ 2.91 (m, 2H), 2.92

(s, 3H), 3.05 (m, 2H), 3.65 (s, 2H), 3.78 (s, 2H), 7.05 (d, J = 8.2 Hz,

1H), 7.18 (d, J = 2.0 Hz, 1H), 7.30 (ddd, J = 7.8, 4.7, and 0.8 Hz, 1H),

7.48−7.38 (m, 3 H), 7.44 (s, 1H), 7.72 (m, 1H), 7.90 (m, 1H), 7.96

(m, 1H), 8.46 (dd, J = 4.9 and 1.8 Hz, 1H), 8.50 (m, 1H). ESIMS m/z

(rel intensity) 386.2 ([M + H], 100).

(R)-4-((9-Chloro-7-(5-ﬂuoro-1H-indol-1-yl)-3-methyl-2,3-

dihydrobenzo[f ][1,4]oxazepin-4(5H)-yl)methyl)pyridin-2(1H)-

one (54). Following the general reductive amination procedure, 96

(10.53 g, 31.8 mmol) and 2-hydroxyisonicotinaldehyde (4.31 g,

35.0 mmol) provided the title compound as a white solid (9.45 g,

1

68%). H NMR (CDCl₃) δ 1.36 (d, J = 6.7 Hz, 3H), 3.36 (m, 1H),

3.55 (d, J = 15.3 Hz, 1H), 3.68 (d, J = 15.3 Hz, 1H), 3.79 (d, J = 15.8

Hz, 1H), 4.08 (dd, J = 12.8 and 6.4 Hz, 1H), 4.28 (m, 2H), 6.43 (br s,

1H), 6.54 (s, 1H), 6.61 (dd, J = 3.3 and 0.6 Hz, 1H), 7.01 (td, J = 9.0

and 2.5 Hz, 1H), 7.03 (s, 1H), 7.28 (m, 2H), 7.33 (d, J = 6.7 Hz, 1H),

7.39 (dd, J = 9.0 and 4.3 Hz, 1H), 7.45 (d, J = 2.5 Hz, 1H). ESIMS

m/z (rel intensity) 438.2 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-

4,5-dihydrobenzo[f ][1,4]oxazepin-3(2H)-one (49). NaBH(OAc)₃

(824 mg, 3.89 mmol) was added to a solution of 87 (960 mg,

2.59 mmol) and pyridin-3-ylmethanamine (290 μL, 2.35 mmol) in

dichloroethane (40 mL) at rt. The reaction mixture was stirred at rt for

2 h, at which point LCMS indicated that the reductive amination was

complete. The reaction mixture was heated at reﬂux for 4.5 h, cooled

to rt, diluted with EtOAc (100 mL), and washed with water (75 mL),

saturated aqueous NaHCO₃, and brine, and dried over MgSO₄,

ﬁltered, and concentrated in vacuo to provide a brown oil. Puriﬁcation

by ﬂash chromatography on silica gel eluting with 2% MeOH/DCM

ASSOCIATED CONTENT

■

S

* Supporting Information

Experimental procedures describing the preparation of

intermediates and protocols for the in vitro assays and phago-

cytosis assay (PDF, CSV). The Supporting Information is

available free of charge on the ACS Publications website at

DOI: 10.1021/acs.jmedchem.5b00567.

1

provided the title compound as an oﬀ-white solid (786 mg, 73%). H

NMR (CDCl₃) δ 3.89 (s, 3H), 4.46 (s, 2H), 4.78 (s, 2H), 4.81 (s, 2H),

6.67 (d, J = 2.0 Hz, 1H), 7.07 (d, J = 2.0 Hz, 1H), 7.22 (s, 1H), 7.24−

7.20 (m, 1H), 7.36 (m, 2H), 7.59 (dt, J = 7.8 and 2.0 Hz, 1H), 7.68

(m, 1H), 7.87 (m, 1H), 8.53 (dd, J = 4.7 and 1.6 Hz, 1H), 8.55 (d, J =

2.0 Hz, 1H). ESIMS m/z (rel intensity) 417.1 ([M + H], 100).

7-(Benzo[b]thiophen-3-yl)-9-methoxy-4-(pyridin-3-ylmethyl)-

3,4-dihydrobenzo[f ][1,4]oxazepin-5(2H)-one (50). NaH (60% in

AUTHOR INFORMATION

■

Corresponding Author

*Phone: (650) 430-3814. E-mail: fox11.brian@gmail.com.

P

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(17) McDonald, D. R.; Brunden, K. R.; Landreth, G. E. Amyloid

fibrils activate tyrosine kinase-dependent signaling and superoxide

production in microglia. J. Neurosci. 1997, 17, 2284−2294.

(18) Lue, L. F.; Walker, D. G.; Rogers, J. Modeling microglial

activation in Alzheimer’s disease with human postmortem microglial

cultures. Neurobiol. Aging 2001, 22, 945−956.

(19) Floden, A. M.; Li, S.; Combs, C. K. β-Amyloid-stimulated

microglia induce neuron death via synergistic stimulation of tumor

necrosis factor α and NMDA receptors. J. Neurosci. 2005, 25, 2566−

2575.

(20) Hickman, S. E.; Allison, E. K.; El Khoury, J. Microglial

dysfunction and defective β-amyloid clearance pathways in aging

Alzheimer’s disease mice. J. Neurosci. 2008, 28, 8354−8360.

(21) Krabbe, G.; Halle, A.; Matyash, V.; Rinnenthal, J. L.; Eom, G.

D.; Bernhardt, U.; Miller, K. R.; Prokop, S.; Kettenmann, H.; Heppner,

F. L. Functional impairment of microglia coincides with beta-amyloid

deposition in mice with Alzheimer-like pathology. PLoS One 2013, 8,

e60921.

(22) Fiala, M.; Lin, J.; Ringman, J.; Kermani-Arab, V.; Tsao, G.; Patel,

A.; Lossinsky, A. S.; Graves, M. C.; Gustavson, A.; Sayre, J.; Sofroni, E.;

Suarez, T.; Chiappelli, F.; Bernard, G. Ineffective phagocytosis of

amyloid-beta by macrophages of Alzheimer’s disease patients. J.

Alzheimer’s Dis. 2005, 7, 221−232.

(23) Mawuenyega, K. G.; Sigurdson, W.; Ovod, V.; Munsell, L.;

Kasten, T.; Morris, J. C.; Yarasheki, K. E.; Bateman, R. J. Decreased

clearance of CNS β-amyloid in Alzheimer’s disease. Science 2010, 330,

1774.

(24) Akiyama, H.; Barger, S.; Barnum, S.; Bradt, B.; Bauer, J.; Cole,

G. M.; Cooper, N. R.; Eikelenboom, P.; Emmerling, M.; Fiebich, B. L.;

Finch, C. E.; Frautschy, S.; Griffin, W. S. T.; Hampel, H.; Hull, M.;

Landreth, G.; Lue, L.-F.; Mrak, R.; Mackenzie, I. R.; McGreer, P. L.;

O’Banion, M. K.; Pachter, J.; Pasinetti, G.; Plata-Salaman, C.; Rogers,

J.; Rydel, R.; Shen, Y.; Streit, W.; Strohmeyer, R.; Tooyoma, I.; Van

Muiswinkel, F. L.; Veerhius, R.; Walker, D.; Webster, S.; Wegrzyniak,

B.; Wenk, G.; Wyss-Coray, T. Inflammation and Alzheimer’s disease.

Neurobiol. Aging 2000, 21, 383−421.

(25) Aronoff, D. M.; Canetti, C.; Peters-Golden, M. Prostaglandin E₂

inhibits alveolar macrophage phagocytosis through an E-prostanoid 2

receptor-mediated increase in intracellular cyclic AMP. J. Immunol.

2004, 173, 559−565.

(26) Montine, T. J.; Sidell, K. R.; Crews, B. C.; Markesbery, W. R.;

Marnett, L. J.; Roberts, L. J., II; Morrow, J. D. Elevated CSF

prostaglandin E₂levels in patients with probable AD. Neurology 1999,

53, 1495−1498.

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, amyloid-beta; IL-1β, interleukin-1β;

IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IL-8,

interleukin-8; MIP-1α, macrophage inﬂammatory protein-1α;

MCP-1, monocyte chemoattractant peptide-1; PGE₂, prosta-

glandin E₂; LPS, lipopolysaccharide; PK, pharmacokinetic

REFERENCES

■

(1) Braak, H.; Braak, E. Frequency of stages of Alzheimer-related

lesions in different age categories. Neurobiol. Aging 1997, 18, 351−357.

(2) Rogers, J.; Strohmeyer, R.; Kovelowski, C. J.; Li, R. Microglia and

inflammatory mechanisms in the clearance of amyloid beta peptide.

Glia 2002, 40, 260−269.

(3) Shie, F.-S.; Breyer, R. M.; Montine, T. J. Microglia lacking E

prostanoid receptor subtype 2 have enhanced Aβ phagocytosis yet lack

Aβ-activated neurotoxicity. Am. J. Pathol. 2005, 166, 1163−1172.

(4) Simard, A. R.; Soulet, D.; Gowing, G.; Julien, J.-P.; Rivest, S. Bone

marrow-derived microglia play a critical role in restricting senile plaque

formation in Alzheimer’s disease. Neuron 2006, 49, 489−502.

(5) Stalder, M.; Phinney, A.; Probst, A.; Sommer, B.; Staufenbiel, M.;

Jucker, M. Association of microglia with amyloid plaques in brains of

APP23 transgenic mice. Am. J. Pathol. 1999, 154, 1673−1684.

(6) Frautschy, S. A.; Yang, F.; Irrizarry, M.; Hyman, B.; Saido, T. C.;

Hsiao, K.; Cole, G. M. Microglial response to amyloid plaques in

APPsw transgenic mice. Am. J. Pathol. 1998, 152, 307−317.

(7) Perlmutter, L. S.; Scott, S. A.; Barron, E.; Chui, H. C. MHC class

II-positive microglia in human brain: association with Alzheimer

lesions. J. Neurosci. Res. 1992, 33, 549−558.

(8) Dickson, D. W.; Farlo, J.; Davies, P.; Crystal, H.; Fuld, P.; Yen, S-

H. C. A double-labeling immunohistochemical study of senile plaques.

Am. J. Pathol. 1988, 132, 86−101.

(9) Itagaki, S.; McGreer, P. L.; Akiyama, H.; Zhu, S.; Selkoe, D.

Relationship of microglia and astrocytes to amyloid deposits of

Alzheimer disease. J. Neuroimmunol. 1989, 24, 173−182.

(10) Sheng, J. G.; Mrak, R. E.; Griffin, W. S. T. Neuritic plaque

evolution in Alzheimer’s disease is accompanied by transition of

activated microglia from primed to enlarged to phagocytic forms. Acta

Neuropathol. 1997, 94, 1−5.

(27) Breyer, R. M.; Bagdassarian, C. K.; Myers, S. A.; Breyer, M. D.

Prostanoid receptors: subtypes and signaling. Annu. Rev. Pharmacol.

Toxicol. 2001, 41, 661−690.

(11) Malm, T. N.; Koistinaho, M.; Parepalo, M.; Vatanen, T.; Ooka,

̈

A.; Karlsson, S.; Koistinaho, J. Bone-marrow-derived cells contribute to

the recruitment of microglial cells in response to beta-amyloid

deposition in APP/PS1 double transgenic Alzheimer mice. Neurobiol.

Dis. 2005, 18, 134−142.

(12) Kalaria, R. N.; Cohen, D. L.; Premkumar, D. R. D. Cellular

aspects of the inflammatory response in Alzheimer’s disease.

Neurodegeneration 1996, 5, 497−503.

(13) Bacskai, B. J.; Kajdasz, S. T.; Christie, R. H.; Carter, C.; Games,

D.; Seubert, P.; Schenk, D.; Hyman, B. T. Imaging of amyloid-β

deposits in brains of living mice permits direct activation of clearance

of plaques with immunotherapy. Nature Med. 2001, 7, 369−372.

(14) Liu, Y.; Walter, S.; Stagi, M.; Cherny, D.; Letiembre, M.; Schulz-

Schaeffer, W.; Heine, H.; Penke, B.; Neumann, H.; Fassbender, K. LPS

receptor (CD14): a receptor for phagocytosis of Alzheimer’s amyloid

peptide. Brain 2005, 128, 1778−1789.

(15) Nicoll, J. A. R.; Wilkinson, D.; Holmes, C.; Steart, P.; Markham,

H.; Weller, R. O. Neuropathology of human Alzheimer disease after

immunization with amyloid-β peptide: a case report. Nature Med.

2003, 9, 448−452.

(16) Kim, S. U.; de Vellis, J. Microglia in health and disease. J.

Neurosci. Res. 2005, 81, 302−313.

(28) Liang, X.; Wang, Q.; Hand, T.; Wu, L.; Breyer, R. M.; Montine,

T. J.; Andreasson, K. Deletion of the prostaglandin E2 EP2 receptor

reduces oxidative damage and amyloid burden in a model of

Alzheimer’s Disease. J. Neurosci. 2005, 25, 10180−10187.

(29) Keene, C. D.; Chang, R. C.; Lopez-Yglesias, A. H.; Shalloway, B.

R.; Sokal, I.; Li, X.; Reed, P. J.; Keene, L. M.; Montine, K. S.; Breyer, R.

M.; Rockhill, J. K.; Montine, T. J. Suppressed accumulation of cerebral

amyloid β peptides in aged transgenic Alzheimer’s disease mice by

transplantation with wild-type or prostaglandin E₂receptor subtype 2-

null bone marrow. Am. J. Pathol. 2010, 177, 346−354.

(30) Johansson, J. U.; Pradhan, S.; Lokteva, L. A.; Woodling, N. S.;

Ko, N.; Brown, H. D.; Wang, Q.; Loh, C.; Cekanaviciute, E.;

Buckwalter, M.; Manning-Bog, A. B.; Andreasson, K. I. Suppression of

inflammation with conditional deletion of the prostanoid E₂EP2

receptor in macrophages and brain microglia. J. Neurosci. 2013, 33,

16016−16032.

(31) Montine, T. J.; Milatovic, D.; Gupta, R. C.; Valyi-Nagy, T.;

Morrow, J. D.; Breyer, R. M. Neuronal oxidative damage from

activated innate immunity is EP₂receptor-dependent. J. Neurochem.

2002, 83, 463−470.

Q

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(32) Shie, F.-S.; Montine, K. S.; Breyer, R. M.; Montine, T. J.

Microglial EP2 is critical to neurotoxicity from activated cerebral

innate immunity. Glia 2005, 52, 70−77.

(33) Johansson, J. U.; Woodling, N. S.; Wang, Q.; Panchal, M.; Liang,

X.; Trueba-Saiz, A.; Brown, H. D.; Mhatre, S. D.; Loui, T.; Andreasson,

K. I. Prostaglandin signaling suppresses beneficial microglial function

in Alzheimer’s disease models. J. Clin. Invest. 2015, 125, 350−364.

(34) Ganesh, T. Prostanoid receptor EP2 as a therapeutic target. J.

Med. Chem. 2014, 4454−4465.

(35) Forselles, K. J.; Root, J.; Clarke, T.; Davey, D.; Aughton, K.;

Dack, K.; Pullen, N. In vitro and in vivo characterization of PF-

04418948, a novel, potent and selective prostaglandin EP₂receptor

antagonist. Br. J. Pharmacol. 2011, 164, 1847−1856.

(36) Birrell, M. A.; Maher, S. A.; Buckley, J.; Dale, N.; Bonvini, S.;

Raemdonck, K.; Pullen, N.; Giembycz, M. A.; Belvisi, M. G. Selectivity

profiling of the novel EP₂receptor antagonist, PF-04418948, in

functional bioassay systems: atypical affinity at the guinea pig EP₂

receptor. Br. J. Pharmacol. 2013, 168, 129−138.

(37) Jiang, J.; Ganesh, T.; Du, Y.; Quan, Y.; Serrano, G.; Qui, M.;

Speigel, I.; Rojas, A.; Lelutiu, N.; Dingeldine, R. Small molecule

antagonist reveals seizure-induced mediation of neuronal injury by

prostaglandin E2 receptor subtype EP2. Proc. Natl. Acad. Sci. U. S. A.

2012, 109, 3149−3154.

(38) Jiang, J.; Quan, Y.; Ganesh, T.; Pouliot, W. A.; Dudek, F. E.;

Dingledine, R. Inhibition of the prostaglandin receptor EP2 following

status epilepticus reduces delayed mortality and brain inflammation.

Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 3591−3596.

(39) Ganesh, T.; Jiang, J.; Shashidharamurthy, R.; Dingeldine, R.

Discovery and characterization of carbamothioylacrylamides as EP2

selective antagonists. ACS Med. Chem. Lett. 2013, 4, 616−621.

(40) Ganesh, T.; Jiang, J.; Dingeldine, R. Development of second

generation EP2 antagonists with high selectivity. Eur. J. Med. Chem.

2014, 82, 521−535.

(41) Ganesh, T.; Jiang, J.; Yang, M.-S.; Dingeldine, R. Lead

optimization studies of cinnamic amide EP2 antagonists. J. Med.

Chem. 2014, 57, 4173−4184.

(42) Rendic, S.; Di Carlo, F. J. Human cytochrome P450 enzymes: a

status report summarizing their reactions, substrates, inducers, and

inhibitors. Drug Metab. Rev. 1997, 29, 413−580.

(43) Riley, R. J.; Parker, A. J.; Trigg, S.; Manners, C. N. Development

of a generalized, quantitative physicochemical model of CYP3A4

inhibition for use in early drug discovery. Pharm. Res. 2001, 18, 652−

655.

(44) Caggiano, A. O.; Kraig, R. P. Prostaglandin E receptor subtypes

in cultured rat microglia and their role in reducing lipopolysaccharide-

induced interleukin-1β production. J. Neurochem. 1999, 72, 565−575.

(45) Hsiao, K.; Chapman, P.; Nilsen, S.; Eckman, C.; Harigaya, Y.;

Younkin, S.; Yang, F.; Cole, G. Correlative memory deficits, Aβ

elevation, and amyloid plaques in transgenic mice. Science 1996, 274,

99−102.

(46) Barder, T. E.; Walker, S. D.; Martinelli, J. R.; Buchwald, S. L.

Catalysts for Suzuki−Miyaura coupling processes: scope and studies of

the effect of ligand structure. J. Am. Chem. Soc. 2005, 127, 4685−4696.

(47) Node, M.; Kumar, K.; Nishide, K.; Ohsugi, S.-I.; Miyamoto, T.

Odorless substitutes for foul-smelling thiols: syntheses and applica-

tions. Tetrahedron Lett. 2001, 42, 9207−9210.

(48) Krow, G. R. Nitrogen insertion reactions of bridged bicyclic

ketones. Regioselective lactam formation. Tetrahedron 1981, 37,

1283−1307.

(49) Antilla, J. C.; Klapars, A.; Buchwald, S. L. The copper-catalyzed

N-arylation of indoles. J. Am. Chem. Soc. 2002, 124, 11684−11688.

(50) Liu, L.; Frohn, M.; Xi, N.; Dominguez, C.; Hungate, R.; Reider,

P. J. A soluble base for the copper-catalyzed imidazole arylations with

aryl halides. J. Org. Chem. 2005, 70, 10135−10138.

R

DOI: 10.1021/acs.jmedchem.5b00567

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

A Selective Prostaglandin E2 Receptor Subtype 2 (EP2) Antagonist Increases the Macrophage-Mediated Clearance of Amyloid-Beta Plaques

DOI: 10.1021/acs.jmedchem.5b00567

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.5b00567

Full text of DOI:10.1021/acs.jmedchem.5b00567

Products guided by the article

R&D Labs maybe for 1093118-59-2

Relevant to this article

Hot Product