Structure-Activity Relationship Study of Heterocyclic Phenylethenyl and Pyridinylethenyl Derivatives as Tau-Imaging Agents That Selectively Detect Neurofibrillary Tangles in Alzheimers Disease Brains

Article

pubs.acs.org/jmc

Structure−Activity Relationship Study of Heterocyclic Phenylethenyl

and Pyridinylethenyl Derivatives as Tau-Imaging Agents That

Selectively Detect Neuroﬁbrillary Tangles in Alzheimer’s Disease

Brains

Kenji Matsumura,^†Masahiro Ono,*^,†Ayane Kitada,^†Hiroyuki Watanabe,^†Masashi Yoshimura,^†

Shimpei Iikuni,^†Hiroyuki Kimura,^†Yoko Okamoto,^‡Masafumi Ihara,^§and Hideo Saji^†

^†Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida

Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan

^‡Department of Pathology, National Cerebral and Cardiovascular Center, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan

^§Department of Stroke and Cerebrovascular Diseases, National Cerebral and Cardiovascular Center, 5-7-1 Fujishiro-dai, Suita, Osaka

565-8565, Japan

S

* Supporting Information

ABSTRACT: In order to explore novel tau-imaging agents

that can selectively detect neuroﬁbrillary tangles in Alzheimer’s

disease (AD) brains, we designed and synthesized a series of

heterocyclic phenylethenyl and (3-pyridinyl)ethenyl deriva-

tives with or without a dimethyl amino group. In in vitro

autoradiography using AD brain sections, all radioiodinated

ligands with a dimethyl amino group bound to Aβ deposits in

the sections. In contrast, the ligands without a dimethyl amino

group showed diﬀerent patterns of radioactivity accumulation

in the sections depending on the kind of heterocycle contained

in their molecules. Particularly, a phenylethenyl benzimidazole

derivative ([¹²⁵I]64) showed marked radioactivity accumu-

lation in the temporal lobe which corresponded with the distribution of tau deposits. [¹²⁵I]64 also showed the most favorable

pharmacokinetics in normal mouse brains (3.69 and 0.06% ID/g at 2 and 60 min postinjection, respectively) among all ligands in

this study. Taken together, these results suggest that [¹²³I]64 may be a new candidate tau-imaging agent.

focused on the development of diagnostic methods for the

disease.

INTRODUCTION

■

Alzheimer’s disease (AD) is a representative neurodegenerative

disease which currently aﬀects millions of elderly people

worldwide. The latest estimates suggest that more than 26.6

million people worldwide suﬀer from AD today, with

predictions that there could be more than 106 million patients

with AD by 2050.¹After the age of 65, the risk of developing

AD doubles every 5 years so that by the age of 85, some recent

studies suggest that ∼50% of individuals will have the disease.²

This means that early therapeutic interventions may lead to the

prevention of irreversible neuronal damage caused by the

disease. However, a deﬁnitive diagnosis still relies on the post-

mortem examination of human brains. Although the mecha-

nism leading to clinical AD symptoms is not completely

understood, the progressive accumulation of senile plaques

(SP) made up of amyloid β peptides (Aβ) and neuroﬁbrillary

tangles (NFT) composed of hyperphosphorylated tau protein

are regarded as two neuropathological hallmarks of the disease.

Since these markers probably appear many years prior to the

cognitive symptoms of AD, detecting Aβ and/or tau in vivo may

lead to an early diagnosis. In recent years, much eﬀort has been

Positron emission tomography (PET) and single photon

emission computed tomography (SPECT) are useful for

imaging Aβ and/or tau noninvasively in living brain tissue.

Since deposits of Aβ have been demonstrated in the early stages

of the disease process,³PET- and SPECT-imaging agents

targeting Aβ, such as [¹¹C]PIB,⁴[¹¹C]SB-13,⁵[¹¹C]BF-227,⁶

[¹⁸F]GE067 (ﬂutemetamol),⁷[¹⁸F]BAY94-9172 (ﬂorbetaben),⁸

[¹⁸F]AV-45 (ﬂorbetapir),⁹

[

¹²³I]IMPY,¹⁰[¹⁸F]AZD4694

([¹⁸F]NAV4694),¹¹and [¹⁸F]FIBT¹²have been developed.

Some of them have been applied for clinical studies and

facilitated the imaging of Aβ in living brains. Since these agents

have enabled us to visualize the deposits of Aβ in AD brains,

which have been conﬁrmed only by the post-mortem

examination so far, they have been used as one of the tools

for the diagnosis of AD.

Received: March 18, 2015

A

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Design strategy for this work (A). Chemical structure of phenylethenyl and (3-pyridinyl)ethenyl benzothiazole (B), benzoxazole (C),

benzofuran (D), benzothiophene (E), and benzimidazole (F) derivatives.

Among these agents, [¹¹C]PIB is considered as one of the

gold standards of Aβ imaging and has been used in many

clinical studies, demonstrating its utility. Moreover, [¹⁸F]AV-45,

[¹⁸F]GE067, and [¹⁸F]BAY94-9172 have been approved by the

U.S. FDA over the past few years and applied to clinical studies

in order to elucidate the relationship between Aβ deposits and

the severity of clinical symptoms of AD. However, some clinical

studies on Aβ imaging using [¹¹C]PIB have shown a poor

association between the density of Aβ deposits and the severity

of dementia or neuronal loss.¹³⁻¹⁵These studies suggest that it

is not possible to diagnose AD with a high level of accuracy

only by Aβ imaging. Meanwhile, many candidates for anti-Aβ

drugs have been developed to reduce the amount of Aβ.^16,17

However, some clinical trials of these drugs have ended in

failure, indicating that other markers expressed downstream of

Aβ deposits may be better suited to evaluate the disease

progression.¹⁸⁻²⁰

The combination of the increased cerebrospinal ﬂuid (CSF)

concentrations of total tau and phosphorylated tau and

decreased concentration of Aβ may act as pathological CSF

biomarker signatures that are diagnostic criteria for AD.²¹

However, lumbar puncture is still considered to be an invasive

procedure for the widespread screening of the population.

Additionally, CSF measurements do not provide information

on regional brain deposits of tau, which may have clear

correlations with cognition or regional brain atrophy, and might

not be able to provide important information regarding the

therapeutic outcomes or response to current drugs aimed at

modulating the amount of tau, such as immunotherapy.

The accumulation of tau occurs in the transentorhinal area,

followed by the involvement of the entorhinal cortex and

hippocampus, progressing to all isocortical association

areas.^22,23The distribution of tau deposits corresponds with

the severity of clinical symptoms of AD.²³⁻²⁶There are several

PET-imaging agents targeting tau, such as [¹⁸F]THK-5105,^27,28

[¹⁸F]THK-5117,^28,29[¹⁸F]T807,^30,31[¹⁸F]T808,^32,33[¹¹C]-

PBB3,^34,35[¹¹C]NML,³⁶[¹⁸F]NML,³⁷and ¹⁸F-labeled or ¹¹C-

labeled astemizole derivatives.³⁸Some of these agents have

been investigated clinically, and their radioactivity accumulation

in living brains corresponded to the distribution of tau deposits

reported previously. In contrast, the approach in developing

SPECT-imaging agents targeting tau in living brains has

markedly lagged behind. Since SPECT imaging has advantages

in terms of the operating cost and already-installed rate in

medical hospitals, it may be more suitable as primary screening

for AD patients than PET imaging. Therefore, further

investigations are needed to develop useful tau-imaging agents

for SPECT. Recently, we reported radioiodinated ligands such

as [¹²⁵I]TH2,³⁹¹²⁵I]3-OI,⁴⁰¹²⁵I]PDB-3,⁴¹and [¹²⁵I]SBIM-3⁴²

[ [

as tau-imaging agents for SPECT. Some in vitro autoradio-

graphic and ﬂuorescent-staining experiments showed the

binding aﬃnity of these agents for tau deposits in AD brain

sections. Among these agents, [¹²⁵I]SBIM-3 (Figure 1A) based

on a benzimidazole scaﬀold showed a higher initial uptake into

(3.28% ID/g at 2 min postinjection) and more rapid clearance

from (0.33% ID/g at 60 min postinjection) normal mouse

brains than any other agent. However, [¹²⁵I]SBIM-3 (or SBIM-

3) also showed marked binding aﬃnity for Aβ in in vitro

autoradiographic and ﬂuorescent-staining experiments using

AD brain sections, indicating that further improvement of the

ligands is necessary to develop more useful tau-imaging agents

which have a higher aﬃnity for tau and lower aﬃnity for Aβ

than SBIM-3. On comparing in vitro autoradiography of

[

¹²⁵I]PDB-3 based on a benzothiazole scaﬀold with that of

¹²⁵I]SBIM-3, we observed some diﬀerences in the distribution

of their radioactivity accumulation in AD brain sections. These

results suggest that the heterocyclic moiety may be involved in

the binding aﬃnity for tau and/or Aβ deposits in AD brains.

In the present study, we planned to replace the

benzimidazole scaﬀold in SBIM-3 with other aromatic hetero-

cycles such as benzothiazole, benzoxazole, benzofuran, and

benzothiophene, and investigated the binding aﬃnity for tau

and Aβ. Additionally, since most Aβ-imaging agents, such as

[¹¹C]PIB, [¹⁸F]GE067, [¹⁸F]BAY94-9172, and [¹⁸F]AV-45,

B

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 1. Synthetic Routes for Phenylethenyl and (3-Pyridinyl)ethenyl Benzothiazole Derivatives (A) and Their Precursors

(B)

Scheme 2. Synthetic Routes for Phenylethenyl (A) and (3-Pyridinyl)ethenyl (B) Benzoxazole Derivatives

contain an amino group in their molecules,⁴⁻¹¹the moiety may

be important for the interaction with Aβ. Therefore, we also

designed and synthesized ligands with or without a dimethyl

amino group in molecules to investigate the eﬀect of the

dimethyl amino group on the binding aﬃnity for tau and Aβ.

Moreover, in our previous studies, we succeeded in improving

the pharmacokinetics in normal mouse brains by exchanging a

phenyl group with a pyridyl group in molecules.^43,44On the

basis of this knowledge, we also planned to substitute a phenyl

group with a pyridyl group in molecules to develop agents

which display more favorable pharmacokinetics in brains.

Consequently, based on the design strategy described above,

we synthesized novel phenylethenyl and (3-pyridinyl)ethenyl

derivatives which contained a diﬀerent type of heterocycle in

their molecules (Figure 1B−F) and investigated the binding

aﬃnity for tau and Aβ deposits in AD brains and

pharmacokinetics in normal mouse brains. To our knowledge,

this is the ﬁrst report on the structure−activity relationship

study focused on the eﬀect of a heterocyclic moiety and

C

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 3. Synthetic Routes for Phenylethenyl and (3-Pyridinyl)ethenyl Benzofuran Derivatives

Scheme 4. Synthetic Routes for Phenylethenyl and (3-Pyridinyl)ethenyl Benzothiophene Derivatives

dimethyl amino group on the selective binding aﬃnity for tau

deposits in AD brains and pharmacokinetics in normal mouse

brains.

condensation reaction between two reagents, as shown in

Schemes 2, 3, and 4, respectively. Then, the corresponding

tributyltin precursors were obtained by the same reaction

described above. These tributyltin precursors were readily

reacted with iodine in chloroform at room temperature to yield

iodo ligands. Phenylethenyl and (3-pyridinyl)ethenyl benzimi-

dazole derivatives (64, 65, 70, 71, 72, and 73) were obtained by

an intermolecular cyclization reaction between 4-iodobenzene-

1,2-diamine or 4-bromobenzene-1,2-diamine with cinnamalde-

hyde, 68, or 69 using Na₂S₂O₅as an oxidant agent. Derivatives

66, 74, and 75 were obtained by protecting the amino groups

of 65, 72, and 73 with a tert-butoxycarbonyl (Boc) group.

Derivatives 67, 76, and 77 were obtained using the same

reaction as described above. Purities of key ligands were shown

to be higher than 95% by high-performance liquid chromatog-

raphy (HPLC).

RESULTS AND DISCUSSION

■

Chemistry. The synthetic routes of heterocyclic phenyl-

ethenyl and (3-pyridinyl)ethenyl derivatives are outlined in

Schemes 1, 2, 3, 4, and 5. Nineteen novel iodinated ligands

(Figure 1B−F) and tributyltin precursors were successfully

prepared according to conventional methods and veriﬁed with

¹H NMR, MS (ESI or APCI), and HRMS (EI).

Phenylethenyl and (3-pyridinyl)ethenyl benzothiazole de-

rivatives (3, 4, 5, 6, 9, 10, 11, and 12) were obtained by a

condensation reaction between each carbaldehyde with 6-

bromo-2-methylbenzothiazole or 6-iodo-2-methylbenzothia-

zole, as shown in Scheme 1. The bromo ligands (9, 10, 11,

and 12) were then reacted with bis(tributyltin) using Pd(0) as

the catalyst, and the corresponding tributyltin precursors (13,

14, 15, and 16) were obtained. Other heterocyclic (benzox-

azole, benzofuran, and benzothiophene) phenylethenyl and (3-

pyridinyl)ethenyl derivatives were also obtained by a

Immunohistochemical Staining Using AD Brain

Sections. After conﬁrmation of the distribution of tau and

Aβ deposits in brain sections from an AD patient by

immunohistochemical staining, we used them for the following

experiments. In our previous studies, we had diﬃculty

D

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 5. Synthetic Routes for Phenylethenyl Benzimidazole Derivatives (A), Their Precursors (B), (3-Pyridinyl)ethenyl

Benzimidazole Derivatives (C), and Their Precursors (D)

evaluating the binding aﬃnity of ligands for tau and Aβ deposits

in AD brains by in vitro autoradiography since we used only

brain sections from the temporal lobe, where both tau and Aβ

accumulated abundantly. To further verify the binding aﬃnity

of the ligands for tau deposits clearly, we needed to carry out

ﬂuorescent-staining experiments by which we could observe

images with a higher resolution than by in vitro auto-

radiography. In the present study, to evaluate the binding

aﬃnity of ligands for tau and Aβ by in vitro autoradiography

more clearly than before, we used AD brain sections from two

diﬀerent regions, the basal frontal and temporal lobes. It is well

known that tau and Aβ accumulate at diﬀerent proportions in

these regions. First, we carried out immunohistochemical

Figure 2. Immunohistochemical staining with antibodies against

staining of tau and Aβ using the sections from the frontal lobe.

Aβ₁₋₄₂(BC05: A and C) and phosphorylated tau (AT8: B and D) in

We conﬁrmed that Aβ accumulated abundantly in the cortex of

sections of the frontal lobe (A and B) and temporal lobe (C and D)

the frontal lobe (Figure 2A), whereas there were no tau

from an AD patient (76-year-old male).

deposits in the same region (Figure 2B). Next, we also carried

out the same staining using the sections from the temporal

lobe. Similarly to the sections from the frontal lobe, we

observed Aβ deposits in the cortex of the temporal lobe (Figure

2C). Although we also observed abundant deposits of tau in the

same region (Figure 2D), the pattern of tau deposits was

completely diﬀerent from that of Aβ deposits. Therefore, we

used these brain sections to determine the selective binding

aﬃnity for tau according to the criteria that favorable tau-

imaging agents should accumulate in the sections from the

temporal lobe, not the frontal lobe. On the basis of the points

described above, we carried out the following in vitro

autoradiographic experiments.

E

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 6. Two Radiolabeling Methods of Heterocyclic Phenylethenyl and (3-Pyridinyl)ethenyl Derivatives

Radiolabeling. In accordance with the solvent and HPLC

conditions as shown in Table S1, we carried out radiolabeling

of each ligand. The radioiodinated ligands with a benzothiazole,

benzoxazole, benzofuran, and benzothiophene moiety were

prepared from the corresponding tributyltin precursors through

an iododestannylation reaction using hydrogen peroxide as an

oxidant with a radiochemical yield of 12−85% (Scheme 6A).

The radioiodinated ligands with a benzimidazole moiety were

prepared from the corresponding tributyltin precursors through

the iododestannylation reaction followed by stirring with TFA

to remove the Boc-protecting groups, with a radiochemical

yield of 25−40% (Scheme 6B). After puriﬁcation by HPLC, the

speciﬁc activity of the no-carrier-added preparation was

comparable to that of [¹²⁵I]NaI (81.4 TBq/mmol). Finally,

the identity of each ligand was veriﬁed with HPLC by

coinjection with a nonradioactive ligand from HPLC data. All

radioiodinated ligands were obtained with a radiochemical

purity of >95% after puriﬁcation by HPLC. It is well known

that phenylethenyl and pyridinylethenyl derivatives show

photoisomerization.^45,46We observed two signal peaks of

almost all heterocyclic phenylethenyl and (3-pyridinyl)ethenyl

derivatives on HPLC analysis, which indicated that these

derivatives showed photoisomerization (Table S1).

First, radioiodinated heterocyclic phenylethenyl and (3-

pyridinyl)ethenyl derivatives with a dimethyl amino group

(including [¹²⁵I]SBIM-3, which we previously reported as a tau-

imaging agent⁴²) were investigated for the binding aﬃnity for

tau and Aβ by in vitro autoradiography using the AD brain

sections, as shown in Figure 3. Figure 3A and B shows the

autoradiographic images of a phenylethenyl benzothiazole

derivative ([¹²⁵I]3). Figure 3C and D shows the autoradio-

graphic images of a (3-pyridinyl)ethenyl benzothiazole

derivative ([¹²⁵I]4). We observed marked radioactivity

In Vitro Autoradiography. In our previous studies, we

carried out binding experiments using recombinant tau

aggregates to quantify the binding aﬃnity of ligands for

tau.^41,42However, a previous study indicated that these

aggregates do not completely imitate the conformation of

native tau deposits in AD brains.⁴⁷Since an assay using human

brain samples from AD patients may provide more reliable

information on the tau−ligand interaction than that using

recombinant tau aggregates, such an experiment is commonly

used in other research.⁴⁸Therefore, in the present study, we

planned to evaluate the binding aﬃnity of ligands for tau based

on in vitro autoradiography using AD brain sections.

Figure 3. Comparison of in vitro autoradiography of [¹²⁵I]3 (A and B),

[

¹²⁵I]4 (C and D), [¹²⁵I]28 (E and F), [¹²⁵I]22 (G and H), [¹²⁵I]43 (I

and J), [¹²⁵I]44 (K and L), [¹²⁵I]59 (M and N), [¹²⁵I]60 (O and P),

¹²⁵I]SBIM-3 (Q and R), and [¹²⁵I]70 (S and T) in brain sections from

[

the AD patient. A, C, E, G, I, K, M, O, Q, and S are the sections from

the temporal lobe. B, D, F, H, J, L, N, P, R, and T are the sections from

the frontal lobe.

F

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

accumulation of [¹²⁵I]3 and [¹²⁵I]4 in the cortexes of both the

frontal and temporal lobes (Figure 3A−D). Since we observed

only Aβ deposits in the frontal lobe (Figure 2A and B), the

radioactivity accumulation of [¹²⁵I]3 and [¹²⁵I]4 in the cortex of

the frontal lobe indicated that these ligands bound to Aβ

strongly (Figure 3B and D). The level of radioactivity

accumulation of both ligands, especially [¹²⁵I]4, in the white

matter of the brain was relatively low, indicating the nonspeciﬁc

binding of these ligands in the AD brain to be low. Ligands

such as [¹²⁵I]3 and [¹²⁵I]4, which have a high binding aﬃnity

for Aβ, strongly bind to Aβ deposits in the temporal lobe. As a

result, the signal from the ligands binding to Aβ was so

overwhelming that the signal from the ligands binding to tau

was obscured (Figure 3A and C). In addition to [¹²⁵I]3 and

[

¹²⁵I]4, other heterocyclic derivatives with a dimethyl amino

group also accumulated in the cortex of the frontal lobe, which

indicated these ligands had marked aﬃnity for Aβ (Figure 3F,

H, J, L, N, P, R, and T). Although we could not clearly evaluate

the binding aﬃnity for tau in the brain sections by in vitro

autoradiography (Figure 3A, C, E, G, I, K, M, O, Q, and S), we

conﬁrmed that these ligands bound to tau by ﬂuorescent-

staining experiments (the ﬂuorescent-staining image using

compound 3 was shown in Figure S1). These results suggest

that heterocyclic phenylethenyl and (3-pyridinyl)ethenyl

derivatives with a dimethyl amino group bind to Aβ, and the

diﬀerence in the heterocyclic moiety in their molecules did not

markedly aﬀect their radioactivity accumulation in the cortex of

the AD brain. In our previous study, we conﬁrmed that

[

¹²⁵I]SBIM-3, a phenylethenyl benzimidazole derivative with a

dimethyl amino group, bound to both tau and Aβ deposits in

AD brain sections.⁴²In the present study, we observed the

reproducibility of the binding of SBIM-3 to these deposits in

another AD patient’s brain.

Figure 4. Comparison of in vitro autoradiography of [¹²⁵I]5 (A and B),

[

¹²⁵I]6 (C and D), [¹²⁵I]23 (E and F), [¹²⁵I]29 (G and H), [¹²⁵I]45 (I

and J), [¹²⁵I]46 (K and L), [¹²⁵I]61 (M and N), [¹²⁵I]62 (O and P),

Radioiodinated heterocyclic phenylethenyl and (3-pyridinyl)-

ethenyl derivatives without a dimethyl amino group were also

analyzed with in vitro autoradiography (Figure 4A−T). Figure

4A and B shows the autoradiographic images of a phenyl-

ethenyl benzothiazole derivative ([¹²⁵I]5). Figure 4C and D

shows the autoradiographic images of a (3-pyridinyl)ethenyl

benzothiazole derivative ([¹²⁵I]6). As well as [¹²⁵I]3 and [¹²⁵I]4,

these images showed marked radioactivity accumulation of

[

¹²⁵I]64 (Q and R), and [¹²⁵I]71 (S and T) in brain sections from the

AD patient. A, C, E, G, I, K, M, O, Q, and S are the sections from the

temporal lobe. B, D, F, H, J, L, N, P, R, and T are the sections from the

frontal lobe. U, V, and W are high magniﬁcation images of Figure 2D,

panel Q from this ﬁgure, and 2C, respectively.

cortex of the frontal lobe (Figure 3R and T), benzimidazole

¹²⁵I]5 and [¹²⁵I]6 in the cortexes of both the frontal and

derivatives without the dimethyl amino group ([¹²⁵I]64 and

[

¹²⁵I]71) did not show marked radioactivity accumulation in

temporal lobes, respectively (Figure 4A−D). Despite the

elimination of a dimethyl amino group, we could not identify

any improvement in the selective binding aﬃnity of these

ligands for tau deposits in the AD brain. These results indicated

that the benzothiazole moiety still had such high binding

aﬃnity for Aβ that the signal from the ligand bound to Aβ still

overwhelmed the signal from the ligand bound to tau. On the

basis of many previous studies regarding Aβ-imaging agents, it

is well known that the substituted amino group plays an

important role in binding to Aβ deposits in AD brains.

However, the results of this experiment also indicated that a

heterocyclic moiety in molecules may be as important to the

binding aﬃnity for Aβ as the substituted amino group. Similarly

to [¹²⁵I]5 and [¹²⁵I]6, other heterocyclic (benzoxazole,

benzofuran, and benzothiophene) derivatives bound to Aβ

(Figure 4F, H, J, L, N, and P). Although we could not identify

any eﬀect of the elimination of a dimethyl amino group from

these ligands, only phenylethenyl and (3-pyridinyl)ethenyl

benzimidazole derivatives showed interesting results. While

benzimidazole derivatives with a dimethyl amino group

([¹²⁵I]SBIM-3 and [¹²⁵I]70) bound to Aβ deposits in the

the cortex of the frontal lobe, where only Aβ accumulated

abundantly (Figure 4R and T). The results indicated that the

binding aﬃnity of [¹²⁵I]64 and [¹²⁵I]71 for Aβ was markedly

decreased because of the elimination of the dimethyl amino

group from [¹²⁵I]SBIM-3 and [¹²⁵I]70. Conversely, radio-

activity accumulation of these ligands in the cortex of the

temporal lobe was maintained, which indicated that these

ligands still had the binding aﬃnity for tau even after the

removal of the dimethyl amino group (Figure 4Q and S). Since

the level of the nonspeciﬁc binding of [¹²⁵I]64 in the white

matter of the brain was lower than that of [¹²⁵I]71, we could

obtain a clear autoradiographic image using [¹²⁵I]64. Compared

to the pattern of radioactivity accumulation of [¹²⁵I]64 in the

cortex of the temporal lobe with that of tau and Aβ deposits,

the radioactivity accumulation of [¹²⁵I]64 (Figure 4V)

corresponded with tau deposits (Figure 4U) more closely

than Aβ deposits (Figure 4W). Moreover, [¹²⁵I]64 showed

almost no accumulation in a control brain section (Figure S2).

Next, we quantitatively evaluated the relationship between

the radioactivity accumulation of [¹²⁵I]64 in the sections and

G

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

the density of tau and Aβ deposits based on the results of in

vitro autoradiography and immunohistochemical staining. The

cortexes of the frontal and temporal lobes are divided into four

(gyrus cinguli, gyrus rectus, gyrus orbitalis, and gyrus frontalis

inferior) and six (gyrus temporalis transversalis, gyrus

temporalis superior, gyrus temporalis medius, gyrus temporalis

inferior, gyrus parahippocampalis, and hippocampus) regions,

respectively. Then, the proportion of tau and Aβ deposits in

each region was calculated, respectively. The radioactivity

accumulation of [¹²⁵I]64 in the cortex of each region was also

quantiﬁed. Finally, we compared the quantiﬁed radioactivity

accumulation of [¹²⁵I]64 with the proportion of tau and Aβ

deposits in each region. As described above, we observed only

Aβ deposits in the frontal lobe (Figure 5a−d, red columns). In

further molecular modiﬁcations of 64 were needed to develop

ligands which show lower nonspeciﬁc binding.

To further investigate the selectivity of these ligands for tau,

we calculated the ratios of the radioactivity accumulation of

each ligand in the cortex of the temporal lobe to that in the

cortex of the frontal lobe (T/F values). Since the level of Aβ

deposits in the frontal lobe is equivalent to that in the temporal

lobe, it should be possible to evaluate the selective binding of

the ligands for tau deposits in the AD brain by calculating of the

T/F values of each ligand. The T/F values of each ligand were

calculated as shown in Table 1. [¹²⁵I]64 displayed the highest

Table 1. Chemical Structure of Heterocyclic Phenylethenyl

and (3-Pyridinyl)ethenyl Derivatives without a Dimethyl

Amino Group and the T/F Values of These Ligands

ligands

X

Y

Z

T/F value

[

¹²⁵I]5

N

C

N

S

C

N

C

N

C

N

C

N

C

N

1.80

1.60

0.87

1.19

2.62

1.36

1.33

1.81

5.85

1.09

[

¹²⁵I]6

S

¹²⁵I]23

¹²⁵I]29

¹²⁵I]45

¹²⁵I]46

¹²⁵I]61

¹²⁵I]62

¹²⁵I]64

¹²⁵I]71

O

Figure 5. Radioactivity of [¹²⁵I]64 that accumulated in the cortexes of

the frontal and temporal lobes (green columns) and the percent areas

of immunostaining for tau (blue columns) and Aβ (red columns) in

the same region. The cortexes of the frontal and temporal lobes were

divided into four (a, b, c, and d) and six (e, f, g, h, i, and j) regions,

respectively. The regions a, b, c, and d are gyrus cinguli, gyrus rectus,

gyrus orbitalis, and gyrus frontalis inferior, respectively. The regions e,

f, g, h, i, and j are gyrus temporalis transversalis, gyrus temporalis

superior, gyrus temporalis medius, gyrus temporalis inferior, gyrus

parahippocampalis, and hippocampus, respectively.

O

S

NH

T/F value (T/F value of [¹²⁵I]64 = 5.85) among all ligands

without a dimethyl amino group (T/F values of the other

ligands = 0.87−2.62), suggesting that [¹²⁵I]64 is a promising

lead ligand for the development of useful tau-imaging agents.

In Vivo Biodistribution in Normal Mice. To evaluate the

uptake of heterocyclic phenylethenyl and (3-pyridinyl)ethenyl

derivatives into brains, a biodistribution study in normal mice

was performed with each ligand, as shown in Table 2 and

Figure S4. Since there are no tau deposits in normal mice, a

high initial uptake and rapid clearance from the brain are

thought to be highly desirable properties of useful tau-imaging

agents. In general, radiotracers with moderate lipophilicity,

indicated by log D_7.4values in the approximate range 2.0−3.5,

show optimal passive brain uptake in vivo.⁵¹Since the log D_7.4-

values of these ligands indicated that these ligands displayed

uptake into normal mouse brains (Table 2). We conﬁrmed that

all ligands displayed uptake into the brains and diﬀerent

pharmacokinetics depending on the kind of heterocycle

contained in the molecule (Table 2). Compared with the

ratios of the uptake of ligands into brains at 2 and 60 min after

injection (brain_{2 min}and brain_{60 min}, respectively), phenylethenyl

the temporal lobe, the proportion of tau deposits was much

higher than that of Aβ deposits (Figure 5e−j, blue and red

columns, respectively). Compared to the calculated radio-

activity accumulation of [¹²⁵I]64 in each region with the

proportion of tau and Aβ deposits in the corresponding region,

the distribution of [¹²⁵I]64 corresponded with that of tau

deposits more closely than that of Aβ deposits (Figure 5a−j,

green columns). Although the radioactivity accumulation of the

other heterocyclic derivatives without a dimethyl amino group

([¹²⁵I]5, [¹²⁵I]6, [¹²⁵I]23, [¹²⁵I]29, [¹²⁵I]45, [¹²⁵I]46, [¹²⁵I]61,

[

¹²⁵I]62, and [¹²⁵I]71) showed poorer correspondence with the

distribution of tau deposits (Figure S3), [¹²⁵I]64 could

selectively detect tau deposits in the AD brain. In a previous

study, a ligand based on a benzimidazole scaﬀold with a diethyl

amino group, BF-126, clearly stained tau deposits in in vitro

ﬂuorescent-staining experiments using AD brain sections.⁴⁹

However, another study also suggested that the ligand has a

suﬃcient aﬃnity to stain Aβ deposits in AD brains depending

on its concentration.⁵⁰Similarly, we conﬁrmed that SBIM-3, a

phenylethenyl benzimidazole derivative with a dimethyl amino

group, had the binding aﬃnity for both tau and Aβ deposits in

the AD brain sections, as shown in Figure 3Q and R. Therefore,

the results of in vitro autoradiography of [¹²⁵I]64, as presented

in Figure 4Q and R, strongly suggested that the higher selective

binding aﬃnity of [¹²⁵I]64 for tau deposits than [¹²⁵I]SBIM-3

should be due to the elimination of the dimethyl amino group.

However, the radioactivity accumulation of [¹²⁵I]64 in the

white matter of the brain was relatively high, indicating that

and (3-pyridinyl)ethenyl benzimidazole derivatives (brain_{2 min}

/

brain_{60 min}= 9.94−61.5) showed the highest ratios, followed in

order by phenylethenyl and (3-pyridinyl)ethenyl benzoxazole

(brain_{2 min}/brain_{60 min}= 1.16−16.5), benzothiazole (brain_{2 min}

/

=

brain_{60 min}= 1.27−12.5), benzofuran (brain_{2 min}/brain_{60 min}

0.90−4.72), and benzothiophene (brain_{2 min}/brain_{60 min}

=

0.70−2.13) derivatives. In particular, the uptake of [¹²⁵I]64,

which showed the selective binding aﬃnity for tau in in vitro

autoradiography, into brains at 2 min after the injection was

3.69% ID/g. Moreover, [¹²⁵I]64 also showed a rapid clearance

from the brain (brain_{60 min}of [¹²⁵I]64 was 0.06% ID/g). As a

H

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Table 2. Brain Uptake of Radioactivity after the Injection of Each Derivative in Normal Mice , Ratio of Radioactivity

Accumulation at 2 and 60 min Postinjection (brain_{2 min}/brain_{60 min}), and log D_7.4Values

time after injection (min)

ligands

2

10

30

60

brain_{2 min}/brain_{60 min}

log D_7.4

b

[

¹²⁵I]3

1.49 (0.18)

2.48 (0.36)

2.48 (0.24)

3.75 (0.40)

2.09 (0.22)

2.88 (0.10)

3.90 (0.17)

5.61 (0.61)

1.05 (0.07)

1.30 (0.15)

1.09 (0.09)

2.88 (0.22)

0.87 (0.07)

1.41 (0.14)

0.98 (0.05)

2.41 (0.10)

3.28 (0.25)

2.85 (0.53)

3.69 (0.23)

3.21 (0.26)

1.72 (0.28)

2.83 (0.20)

2.78 (0.12)

2.15 (0.11)

2.01 (0.09)

3.20 (0.21)

3.32 (0.19)

2.87 (0.31)

1.15 (0.05)

1.58 (0.16)

1.47 (0.12)

2.53 (0.17)

0.84 (0.09)

1.86 (0.11)

1.16 (0.09)

2.56 (0.23)

1.61 (0.18)

1.22 (0.06)

1.20 (0.08)

0.61 (0.08)

1.79 (0.22)

1.96 (0.18)

2.34 (0.27)

0.68 (0.07)

2.08 (0.16)

1.93 (0.28)

2.67 (0.27)

0.78 (0.10)

1.10 (0.12)

1.42 (0.09)

1.44 (0.13)

1.38 (0.25)

0.93 (0.08)

1.47 (0.07)

1.31 (0.04)

1.81 (0.13)

0.68 (0.11)

0.26 (0.04)

0.18 (0.02)

0.12 (0.01)

1.41 (0.35)

1.21 (0.27)

1.58 (0.19)

0.30 (0.07)

1.80 (0.14)

1.10 (0.08)

1.75 (0.18)

0.34 (0.05)

1.17 (0.11)

1.07 (0.21)

1.40 (0.14)

0.61 (0.11)

0.88 (0.09)

1.13 (0.09)

1.40 (0.05)

1.20 (0.13)

0.33 (0.06)

0.08 (0.01)

0.06 (0.01)

1.27

3.15

3.25

3.95

3.32

3.37

3.20

3.95

3.35

2.11

2.52

2.38

3.13

2.40

2.85

2.34

2.95

2.23

3.11

3.25

3.15

c

¹²⁵I]4

2.34

c

¹²⁵I]5

¹²⁵I]6

1.76

12.5

1.16

¹²⁵I]28

¹²⁵I]22

¹²⁵I]23

¹²⁵I]29

¹²⁵I]43

¹²⁵I]44

¹²⁵I]45

¹²⁵I]46

¹²⁵I]59

¹²⁵I]60

¹²⁵I]61

¹²⁵I]62

¹²⁵I]SBIM-3

¹²⁵I]70

¹²⁵I]64

¹²⁵I]71

c

2.91

2.23

16.5

0.90

c

1.48

c

1.05

4.72

b

1.06

c

1.65

0.70

c

2.13

d

9.94

35.6

61.5

53.5

a

b

c

Each value is expressed as % ID/g and represents the mean (SD) of 5 animals. Expressed as the brain_{30 min}/brain_{60 min}ratio. Expressed as the

d

brain_{10 min}/brain_{60 min}ratio. Data are from ref 42.

result, the brain_{2 min}/brain_{60 min}ratio of [¹²⁵I]64 was 61.5,

equivalent to or higher than those of [¹⁸F]THK-5105,²⁸

[¹⁸F]THK-5117,²⁸[¹⁸F]T807,³⁰[¹⁸F]T808,³³and [¹¹C]PBB3³⁵

reported previously as tau-imaging agents (9.20, 23.3, 7.17 (the

as the liver and kidney. When we carried out HPLC, the

analysis of the radioactivity in the brain at 2 min postinjection

of [¹²⁵I]64, approximately 92% of [¹²⁵I]64 existed as an intact

form in the brain (Figure S7). This means that the intact [¹²⁵I]

64 can enter the brain, and its radiometabolites hardly enter the

brain despite its relatively rapid metabolism of [¹²⁵I]64 in

plasma.

ratio of brain_{5 min}/brain_{30 min}), 2.91 (the ratio of brain_{2.5 min}

/

brain_{20 min}), and 64.0, respectively). [¹²⁵I]64 displayed a high

uptake in the liver (liver_{2 min}= 17.1% ID/g) and kidney

(kidney_{2 min}= 12.3% ID/g) followed by a rapid clearance

(liver_{60 min}= 2.65% ID/g and kidney_{60 min}= 2.30% ID/g), while

the intestine exhibited an accumulation of radioactivity over

time (intestine_{60 min}= 30.6% ID/g). The accumulation in the

thyroid (thyroid_{60 min}= 0.11% ID) suggested that [¹²⁵I]64 may

be stable for deiodination in vivo by 60 min after the injection

(Table S2). On the basis of in vitro autoradiography using the

AD brain sections and in vivo biodistribution in normal mice for

these series of compounds, [¹²⁵I]64, which exhibits the highest

selective binding aﬃnity for tau and the most favorable

pharmacokinetics in normal mouse brains, was selected for

further biological evaluation.

CONCLUSIONS

■

We designed and synthesized a series of heterocyclic phenyl-

ethenyl and (3-pyridinyl)ethenyl derivatives with or without a

dimethyl amino group to evaluate which moiety plays an

important role in the binding aﬃnity for Aβ and tau deposits in

AD brains. Radioiodinated ligands were synthesized with a

radiochemical yield of 12−85%, and HPLC analysis indicated

that almost all ligands showed photoisomerization. In in vitro

autoradiography using AD brain sections, all heterocyclic

phenylethenyl and (3-pyridinyl)ethenyl derivatives with a

dimethyl amino group showed a high binding aﬃnity for Aβ

in the AD brain sections. In contrast, the derivatives without a

dimethyl amino group showed diﬀerent patterns of radioactivity

accumulation in the cortex of the AD brain depending on the

kind of heterocycle contained in their molecules. In particular,

the radioiodinated phenylethenyl benzimidazole derivative

([¹²⁵I]64) showed marked radioactivity accumulation in the

cortex of the temporal lobe from the AD patient, and the

distribution of radioactivity corresponded with tau deposits,

although there was a relatively high nonspeciﬁc binding of

We determined the in vitro stability of [¹²⁵I]64 in mouse

plasma. After the incubation of [¹²⁵I]64 in mouse plasma for 60

min, the sample was analyzed by HPLC. Since only

radioactivity derived from [¹²⁵I]64 was detected, [¹²⁵I]64

would be stable in mouse plasma until 60 min (Figure S5).

Conversely, when radioactivity was analyzed in mouse plasma

by HPLC after the injection of [¹²⁵I]64 into normal mice, we

identiﬁed the conversion of [¹²⁵I]64 to several diﬀerent

chemical forms (Figure S6). The percent of the intact form

decreased with time and reached 46.5 and 13.1% at 2 and 10

min after injection, respectively. This proﬁle was comparable to

that of [¹⁸F]ﬂutemetamol in rats reported previously,⁵²

suggesting that [^123/125I]64 has suﬃcient in vivo stability for

clinical trials. The diﬀerence in the stability of [¹²⁵I]64 between

in vitro and in vivo may be attributable to radiometabolites

produced by the in vivo metabolism of [¹²⁵I]64 in organs such

[

¹²⁵I]64 in the white matter of the brain. On comparing the

ratios of the radioactivity accumulation of these ligands in the

temporal lobe to the frontal lobe (T/F values), we found that

[

¹²⁵I]64 may detect tau deposits in the AD brain more

selectively than any other ligand (the T/F value of [¹²⁵I]64 =

5.85). In biodistribution experiments using normal mice, [¹²⁵I]

64 showed a high initial uptake into (3.69% ID/g at 2 min

I

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

7.39−7.47 (m, 3H). HRMS (EI) m/z calcd. for C₁₅H₁₀INS (M⁺)

362.9579, found 362.9586.

(E)-6-Iodo-2-(2-(pyridin-3-yl)vinyl)benzo[d]thiazole (6). The same

postinjection) and rapid clearance from (0.06% ID/g at 60 min

postinjection) the brains. In conclusion, these results suggest

that [¹²³I]64 may become a useful tau-imaging agent. Also,

these new ﬁndings of the present study may provide valuable

information for the development of more useful agents that can

selectively detect NFT in AD brains.

reaction described above for the preparation of 3 was employed to

1

aﬀord 30.0 mg of 6 (45.4%). H NMR (400 MHz, DMSO-d₆) δ 8.94

(d, J = 2.0 Hz, 1H), 8.57 (d, J = 1.7 Hz, 2H), 8.24 (d, J = 7.8 Hz, 1H),

7.71−7.84 (m, 4H), 7.47 (dd, J = 8.1, 4.9 Hz, 1H). HRMS (EI) m/z

calcd. for C₁₄H₉IN₂S (M⁺) 363.9531, found 363.9536.

EXPERIMENTAL SECTION

N-(4-Bromo-2-iodophenyl)acetamide (7). The same reaction

■

described above for the preparation of 1 was employed to aﬀord

General Remarks. All reagents were commercial products and

used without further puriﬁcation unless indicated otherwise. All ligands

were puriﬁed by Smart Flash EPCLC W-Prep 2XY (Yamazen

corporation) unless indicated otherwise. ¹H NMR spectra were

recorded on a JNM-ECS400 (JEOL) with TMS as an internal

standard. Coupling constants are reported in Hertz. Multiplicity was

deﬁned as singlet (s), doublet (d), triplet (t), quartet (q), or multiplet

(m). ESI or APCI mass spectrometry was conducted with a

SHIMADZU LCMS-2020. High-resolution mass spectrometry

(HRMS) was carried out with a GCMate II (JEOL). HPLC was

performed with a Shimadzu system (an LC-20AD pump with an SPD-

20AUV detector, λ = 254 nm) using a Cosmosil C₁₈column (Nacalai

Tesque, COSMOSIL 5C₁₈-AR-II 4.6 mm I.D. × 150 mm or

COSMOSIL 5C₁₈-MS-II 4.6 mm I.D. × 150 mm) and acetonitrile/

H₂O as the mobile phase at a ﬂow rate of 1.0 mL/min. All key ligands

were proven by this method to show >95% purity.

1

1.50 g of 7 (88.5%). H NMR (400 MHz, CDCl₃) δ 8.12 (d, J = 8.4

Hz, 1H), 7.90 (d, J = 2.0 Hz, 1H), 7.45 (dd, J = 9.0, 2.3 Hz, 1H), 7.38

(s, 1H), 2.24 (s, 3H).

6-Bromo-2-methylbenzo[d]thiazole (8). The same reaction

described above for the preparation of 2 was employed to aﬀord

134 mg of 8 (29.5%). ¹H NMR (400 MHz, CDCl₃) δ 7.95 (d, J = 1.7

Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.54 (dd, J = 8.7, 2.0 Hz, 1H), 2.82

(s, 3H). MS (ESI) m/z 228.0 [MH⁺].

(E)-4-(2-(6-Bromobenzo[d]thiazol-2-yl)vinyl)-N,N-dimethylaniline

(9). The same reaction described above for the preparation of 3 was

employed to aﬀord 179 mg of 9 (50.0%). ¹H NMR (400 MHz,

CDCl₃) δ 7.94 (d, J = 1.7 Hz, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.52 (dd,

J = 8.7, 2.0 Hz, 1H), 7.46 (d, J = 9.0 Hz, 2H), 7.44 (d, J = 16.0 Hz,

1H), 7.15 (d, J = 16.0 Hz, 1H), 6.71 (d, J = 8.7 Hz, 2H), 3.03 (s, 6H).

MS (ESI) m/z 359.1 [MH⁺].

(E)-5-(2-(6-Bromobenzo[d]thiazol-2-yl)vinyl)-N,N-dimethylpyri-

Chemistry. N-(2,4-Diiodophenyl)acetamide (1). A mixture of 2,4-

diiodoaniline (1.00 g, 2.90 mmol) and acetic anhydride (274 μL, 2.90

mmol) in dichloromethane (10.0 mL) was stirred at room temperature

for 12 h. After pouring it into water, the mixture was extracted with

chloroform (50.0 mL × 2). Combined organic layers were dried over

MgSO₄and concentrated under vacuum. The residue was puriﬁed by

silica gel chromatography (ethyl acetate/hexane = 1/1) to yield 786

din-2-amine (10). The same reaction described above for the

1

preparation of 3 was employed to aﬀord 150 mg of 10 (41.8%). H

NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H), 7.95 (s, 1H), 7.78 (d, J = 8.4

Hz, 1H), 7.70 (dd, J = 9.0, 2.3 Hz, 1H), 7.53 (dd, J = 8.7, 2.0 Hz, 1H),

7.40 (d, J = 16.0 Hz, 1H), 7.13 (d, J = 16.0 Hz, 1H), 6.55 (d, J = 9.0

Hz, 1H), 3.15 (s, 6H). MS (ESI) m/z 360.1 [MH⁺].

(E)-6-Bromo-2-styrylbenzo[d]thiazole (11). The same reaction

described above for the preparation of 3 was employed to aﬀord

50.0 mg of 11 (31.8%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.41 (d, J =

1.4 Hz, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.79 (d, J = 8.0 Hz, 2H), 7.71

(d, J = 16.3 Hz, 1H), 7.65−7.68 (m, 1H), 7.63 (d, J = 16.3 Hz, 1H),

7.39−7.47 (m, 3H). MS (ESI) m/z 316.1 [MH⁺].

1

mg of 1 (70.0%). H NMR (400 MHz, CDCl₃) δ 8.08 (s, 1H), 8.01

(d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.38 (s, 1H), 2.23 (s,

3H).

6-Iodo-2-methylbenzo[d]thiazole (2). A mixture of CuI (42.0 mg,

0.220 mmol), 1 (876 mg, 2.20 mmol), and Na₂S·9H₂O (1.59 g, 6.60

mmol) in DMF (4.00 mL) was stirred at 80 °C for 12 h. The reaction

mixture was cooled to room temperature, and then 2.00 mL of

concentrated HCl was added, and the mixture was stirred at 80 °C for

12 h. After adding 20.0 mL of saturated NaHCO₃aq, it was extracted

with ethyl acetate (50.0 mL × 2). Combined organic layers were dried

over MgSO₄and concentrated under vacuum. The residue was

puriﬁed by silica gel chromatography (ethyl acetate/hexane = 1/4) to

yield 302 mg of 2 (50.0%). ¹H NMR (400 MHz, CDCl₃) δ 8.16 (d, J =

1.5 Hz, 1H), 7.73 (dd, J = 8.7, 1.7 Hz, 1H), 7.68 (d, J = 8.7 Hz, 1H),

2.82 (s, 3H). MS (ESI) m/z 276.0 [MH⁺].

(E)-4-(2-(6-Iodobenzo[d]thiazol-2-yl)vinyl)-N,N-dimethylaniline

(3). To a solution of 2 (275 mg, 1.00 mmol) in MeOH (5.00 mL)

were added p-dimethylaminobenzaldehyde (149 mg, 1.00 mmol) and

solid NaOH (120 mg, 3.00 mmol) as a base catalyst. The reaction

mixture was stirred and heated to reﬂux. After reﬂuxing for 24 h, the

resulting yellow solid was collected by ﬁltration and washed with

MeOH and water. The solid was dried under vacuum to yield 203 mg

of 3 (50.0%). ¹H NMR (400 MHz, CDCl₃) δ 8.14 (d, J = 1.8 Hz, 1H),

7.71 (dd, J = 8.7, 1.6 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.47 (d, J = 8.9

Hz, 2H), 7.46 (d, J = 16.3 Hz, 1H), 7.16 (d, J = 16.3 Hz, 1H), 6.72 (d,

J = 8.9 Hz, 2H), 3.04 (s, 6H). HRMS (EI) m/z calcd. for C₁₇H₁₅IN₂S

(M⁺) 406.0001, found 405.9998.

(E)-6-Bromo-2-(2-(pyridin-3-yl)vinyl)benzo[d]thiazole (12). The

same reaction described above for the preparation of 3 was employed

1

to aﬀord 49.0 mg of 12 (31.0%). H NMR (400 MHz, DMSO-d₆) δ

8.95 (d, J = 2.0 Hz, 1H), 8.57 (d, J = 4.9 Hz, 1H), 8.44 (d, J = 2.0 Hz,

1H), 8.25 (d, J = 7.8 Hz, 1H), 7.93 (d, J = 8.7 Hz, 1H), 7.79 (d, J =

16.5 Hz, 1H), 7.74 (d, J = 16.2 Hz, 1H), 7.67−7.70 (m, 1H), 7.48 (dd,

J = 7.8, 4.6 Hz, 1H). MS (ESI) m/z 317.1 [MH⁺].

(E)-N,N-Dimethyl-4-(2-(6-(tributylstannyl)benzo[d]thiazol-2-yl)-

vinyl)aniline (13). A mixture of 9 (130 mg, 0.360 mmol), (SnBu₃)₂

(364 μL, 0.730 mmol), and (Ph₃P)₄Pd (180 mg, 0.116 mmol) in a

mixed solvent (15.0 mL, dioxane/Et₃N = 2/1) was stirred and heated

to reﬂux. After reﬂuxing for 8 h, the mixture was concentrated under

vacuum, and the residue was puriﬁed by silica gel chromatography

(ethyl acetate/hexane = 1/4) to yield 104 mg of 13 (50.0%). ¹H NMR

(400 MHz, CDCl₃) δ 7.89−7.91 (m, 2H), 7.50 (dd, J = 8.1, 0.9 Hz,

1H), 7.47 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 15.9 Hz, 1H), 7.20 (d, J =

16.0 Hz, 1H), 6.71 (d, J = 9.0 Hz, 2H), 3.02 (s, 6H), 0.87−1.59 (m,

27H). MS (ESI) m/z 571.4 [MH⁺].

(E)-N,N-Dimethyl-5-(2-(6-(tributylstannyl)benzo[d]thiazol-2-yl)-

vinyl)pyridin-2-amine (14). The same reaction described above for

the preparation of 13 was employed to aﬀord 170 mg of 14 (48.4%).

¹H NMR (400 MHz, CDCl₃) δ 8.30 (d, J = 2.3 Hz, 1H), 7.90−7.92

(E)-5-(2-(6-Iodobenzo[d]thiazol-2-yl)vinyl)-N,N-dimethylpyridin-

2-amine (4). The same reaction described above for the preparation of

3 was employed to aﬀord 60.0 mg of 4 (29.5%). ¹H NMR (400 MHz,

CDCl₃) δ 8.31 (d, J = 2.3 Hz, 1H), 8.15 (d, J = 1.7 Hz, 1H), 7.70−7.73

(m, 2H), 7.66 (d, J = 8.7 Hz, 1H), 7.42 (d, J = 16.2 Hz, 1H), 7.13 (d, J

= 16.2 Hz, 1H), 6.56 (d, J = 9.0 Hz, 1H), 3.16 (s, 6H). HRMS (EI) m/

z calcd. for C₁₆H₁₄IN₃S (M⁺) 406.9953, found 406.9955.

(m, 2H), 7.70 (dd, J = 9.0, 2.3 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.40

(d, J = 16.0 Hz, 1H), 7.17 (d, J = 16.0 Hz, 1H), 6.53 (d, J = 9.0 Hz,

1H), 3.12 (s, 6H), 0.88−1.65 (m, 27H). MS (ESI) m/z 572.4 [MH⁺].

(E)-2-Styryl-6-(tributylstannyl)benzo[d]thiazole (15). The same

reaction described above for the preparation of 13 was employed to

1

aﬀord 89.0 mg of 15 (30.1%). H NMR (400 MHz, CDCl₃) δ 7.94−

(E)-6-Iodo-2-styrylbenzo[d]thiazole (5). The same reaction de-

7.97 (m, 2H), 7.33−7.59 (m, 8H), 0.88−1.61 (m, 27H). MS (ESI) m/

z 528.3 [MH⁺].

scribed above for the preparation of 3 was employed to aﬀord 10.0 mg

1

of 5 (15.2%). H NMR (400 MHz, DMSO-d₆) δ 8.55 (s, 1H), 7.75−

(E)-2-(2-(Pyridin-3-yl)vinyl)-6-(tributylstannyl)benzo[d]thiazole

(16). The same reaction described above for the preparation of 13 was

7.82 (m, 4H), 7.70 (d, J = 16.3 Hz, 1H), 7.62 (d, J = 16.3 Hz, 1H),

J

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

employed to aﬀord 76.0 mg of 16 (45.5%). H NMR (400 MHz,

CDCl₃) δ 8.80 (s, 1H), 8.58 (d, J = 4.6 Hz, 1H), 7.89−7.99 (m, 3H),

7.45−7.58 (m, 3H), 7.34 (dd, J = 8.1, 4.9 Hz, 1H), 0.88−1.66 (m,

27H). MS (ESI) m/z 529.3 [MH⁺].

(E)-4-(2-(6-Bromobenzo[d]oxazol-2-yl)vinyl)-N,N-dimethylaniline

(24). To a mixture of 2-amino-5-bromophenol (935 mg, 5.00 mmol)

and 4-(dimethylamino) cinnamic acid (955 mg, 5.00 mmol) was

added polyphosphoric acid (12.0 g). The mixture was stirred at 180 °C

for 1 h. After cooling to room temperature, water (100 mL) was added

to the mixture, and it was neutralized by K₂CO₃. The mixture was

extracted with ethyl acetate (50.0 mL × 2). Combined organic layers

were dried over MgSO₄and concentrated under vacuum. The residue

was puriﬁed by silica gel chromatography (ethyl acetate/hexane = 1/1)

6-Bromo-2-methylbenzo[d]oxazole (17). To a mixture of triethyl

orthoacetate (656 μL, 3.60 mmol) and 2-amino-5-bromophenol (561

mg, 3.00 mmol) was added a catalytic amount of 1,3-dibromo-5,5-

dimethylhydantoin (17.2 mg, 6.00 × 10⁻²mmol). The mixture was

stirred at room temperature for 10 min. The mixture was then

extracted with ethyl acetate (50.0 mL × 2). Combined organic layers

were dried over MgSO₄and concentrated under vacuum. The residue

was puriﬁed by silica gel chromatography (ethyl acetate/hexane = 1/4)

1

to yield 130 mg of 24 (7.60%). H NMR (400 MHz, CDCl₃) δ 7.71

(d, J = 16.2 Hz, 1H), 7.63 (s, 1H), 7.47−7.52 (m, 3H), 7.40 (dd, J =

8.4, 1.7 Hz, 1H), 6.79 (d, J = 16.2 Hz, 1H), 6.70 (d, J = 9.0 Hz, 2H),

3.03 (s, 6H). MS (ESI) m/z 343.2 [MH⁺].

1

to yield 560 mg of 17 (88.5%). H NMR (400 MHz, CDCl₃) δ 7.61

(E)-6-Bromo-2-(2-(pyridin-3-yl)vinyl)benzo[d]oxazole (25). The

same reaction described above for the preparation of 24 was employed

to aﬀord 1.40 g of 25 (93.3%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.98

(s, 1H), 8.60 (d, J = 4.6 Hz, 1H), 8.30 (d, J = 7.8 Hz, 1H), 8.08 (d, J =

1.5 Hz, 1H), 7.87 (d, J = 16.5 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.58

(dd, J = 8.4, 1.7 Hz, 1H), 7.47−7.57 (m, 2H). MS (ESI) m/z 301.1

[MH⁺].

(E)-N,N-Dimethyl-4-(2-(6-(tributylstannyl)benzo[d]oxazol-2-yl)-

vinyl)aniline (26). The same reaction described above for the

preparation of 20 was employed to aﬀord 30.0 mg of 26 (14.2%).

¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J = 16.2 Hz, 1H), 7.65 (d, J =

(d, J = 1.7 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.40 (dd, J = 7.8, 1.2 Hz,

1H), 2.61 (s, 3H). MS (ESI) m/z 212.0 [MH⁺].

(E)-5-(2-(6-Bromobenzo[d]oxazol-2-yl)vinyl)-N,N-dimethylpyri-

din-2-amine (18). To a solution of 17 (560 mg, 2.65 mmol) in MeOH

(5.00 mL) were added 6-(dimethylamino)nicotinaldehyde (398 mg,

2.65 mmol) and solid NaOH (319 mg, 7.96 mmol) as a base catalyst.

The reaction mixture was stirred and heated to reﬂux. After reﬂuxing

for 2.5 h, the resulting yellow solid was collected by ﬁltration and

washed with MeOH and water. The solid was dried under vacuum to

yield 220 mg of 18 (24.1%). ¹H NMR (400 MHz, CDCl₃) δ 8.33 (d, J

= 2.3 Hz, 1H), 7.74 (dd, J = 9.0, 2.3 Hz, 1H), 7.69 (d, J = 16.2 Hz,

1H), 7.66 (d, J = 1.7 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.43 (dd, J =

8.4, 1.7 Hz, 1H), 6.79 (d, J = 16.2 Hz, 1H), 6.57 (d, J = 9.0 Hz, 1H),

3.17 (s, 6H). MS (ESI) m/z 344.1 [MH⁺].

7.5 Hz, 1H), 7.59 (s, 1H), 7.49 (d, J = 9.0 Hz, 2H), 7.36 (d, J = 7.5 Hz,

1H), 6.85 (d, J = 16.0 Hz, 1H), 6.71 (d, J = 8.7 Hz, 2H), 3.03 (s, 6H),

0.88−1.58 (m, 27H). MS (ESI) m/z 555.4 [MH⁺].

(E)-6-Bromo-2-styrylbenzo[d]oxazole (19). The same reaction

described above for the preparation of 18 was employed to aﬀord

235 mg of 19 (18.6%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.05 (d, J =

2.0 Hz, 1H), 7.82−7.86 (m, 3H), 7.71 (d, J = 8.4 Hz, 1H), 7.56 (dd, J

= 8.4, 1.7 Hz, 1H), 7.45−7.47 (m, 3H), 7.34 (d, J = 16.2 Hz, 1H). MS

(ESI) m/z 300.1 [MH⁺].

(E)-2-(2-(Pyridin-3-yl)vinyl)-6-(tributylstannyl)benzo[d]oxazole

(27). The same reaction described above for the preparation of 20 was

employed to aﬀord 826 mg of 27 (40.1%). ¹H NMR (400 MHz,

CDCl₃) δ 8.80 (s, 1H), 8.58 (d, J = 4.9 Hz, 1H), 7.87 (d, J = 7.8 Hz,

1H), 7.66−7.76 (m, 3H), 7.43 (d, J = 7.5, 1H), 7.29−7.32 (m, 1H),

7.15 (dd, J = 16.2, 1.4 Hz, 1H), 0.99−1.62 (m, 27H). MS (ESI) m/z

513.4 [MH⁺].

(E)-N,N-Dimethyl-5-(2-(6-(tributylstannyl)benzo[d]oxazol-2-yl)-

vinyl)pyridin-2-amine (20). A mixture of 18 (100 mg, 0.290 mmol),

(SnBu₃)₂(292 μL, 0.580 mmol), and (Ph₃P)₄Pd (145 mg, 0.130

mmol) in a mixed solvent (15.0 mL, dioxane/Et₃N = 2/1) was stirred

and heated to reﬂux. After reﬂuxing for 4 h, the mixture was

concentrated under vacuum, and the residue was puriﬁed by silica gel

chromatography (ethyl acetate/hexane = 1/4) to yield 74.0 mg of 20

(45.7%). ¹H NMR (400 MHz, CDCl₃) δ 8.32 (d, J = 2.0 Hz, 1H), 7.74

(dd, J = 9.0, 2.3 Hz, 1H), 7.64−7.70 (m, 2H), 7.61 (s, 1H), 7.37 (d, J =

7.5 Hz, 1H), 6.83 (d, J = 16.2 Hz, 1H), 6.56 (d, J = 9.0 Hz, 1H), 3.15

(s, 6H), 0.88−1.77 (m, 27H). MS (ESI) m/z 556.4 [MH⁺].

(E)-2-Styryl-6-(tributylstannyl)benzo[d]oxazole (21). The same

reaction described above for the preparation of 20 was employed to

aﬀord 155 mg of 21 (38.6%). ¹H NMR (400 MHz, CDCl₃) δ 7.78 (d,

J = 16.2 Hz, 1H), 7.70 (d, J = 7.5 Hz, 1H), 7.64 (s, 1H), 7.59 (d, J =

7.3 Hz, 2H), 7.36−7.42 (m, 4H), 7.09 (d, J = 16.5 Hz, 1H), 0.8−1.61

(m, 27H). MS (ESI) m/z 512.4 [MH⁺].

(E)-5-(2-(6-Iodobenzo[d]oxazol-2-yl)vinyl)-N,N-dimethylpyridin-

2-amine (22). To a solution of 20 (70.0 mg, 0.130 mmol) in

chloroform (30.0 mL) was added a solution of iodine in chloroform

(3.00 mL, 50.0 mg/mL). The mixture was stirred at room temperature

for 1 h and saturated NaHSO₃aq was added to quench the reaction.

The mixture was extracted with chloroform (50.0 mL × 2). Combined

organic layers were dried over MgSO₄and concentrated under

vacuum. The residue was puriﬁed by silica gel chromatography (ethyl

acetate/hexane = 1/4) to yield 15.0 mg of 22 (30.4%). ¹H NMR (400

MHz, CDCl₃) δ 8.33 (d, J = 2.3 Hz, 1H), 7.84 (d, J = 1.5 Hz, 1H),

7.73 (dd, J = 9.0, 2.6 Hz, 1H), 7.69 (d, J = 16.2 Hz, 1H), 7.61 (dd, J =

8.4, 1.5 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 16.0 Hz, 1H),

6.56 (d, J = 9.0 Hz, 1H), 3.16 (s, 6H). HRMS (EI) m/z calcd. for

C₁₆H₁₄IN₃O (M⁺) 391.01819, found 391.01764.

(E)-4-(2-(6-Iodobenzo[d]oxazol-2-yl)vinyl)-N,N-dimethylaniline

(28). The same reaction described above for the preparation of 22 was

1

employed to aﬀord 10.0 mg of 28 (50.0%). H NMR (400 MHz,

CDCl₃) δ 7.83 (s, 1H), 7.72 (d, J = 16.0 Hz, 1H), 7.59 (dd, J = 8.1, 1.5

Hz, 1H), 7.49 (d, J = 9.0 Hz, 2H), 7.40 (d, J = 8.4 Hz, 1H), 6.80 (d, J

= 16.0 Hz, 1H), 6.71 (d, J = 9.0 Hz, 2H), 3.04 (s, 6H). HRMS (EI) m/

z calcd. for C₁₇H₁₅IN₂O (M⁺) 390.0229, found 390.0227.

(E)-6-Iodo-2-(2-(pyridin-3-yl)vinyl)benzo[d]oxazole (29). The

same reaction described above for the preparation of 22 was employed

to aﬀord 80.6 mg of 29 (46.3%). ¹H NMR (400 MHz, CDCl₃) δ 8.82

(d, J = 2.0 Hz, 1H), 8.62 (dd, J = 4.9, 1.4 Hz, 1H), 7.91−7.94 (m, 2H),

7.78 (d, J = 16.5 Hz, 1H), 7.67 (dd, J = 8.4, 1.5 Hz, 1H), 7.48 (d, J =

8.4 Hz, 1H), 7.38 (dd, J = 7.8, 4.6 Hz, 1H), 7.12 (d, J = 16.5 Hz, 1H).

HRMS (EI) m/z calcd. for C₁₄H₉IN₂O (M⁺) 347.9760, found

347.9754.

4-Bromo-2-hydroxybenzaldehyde (30). 4-Bromophenol (6.50 mL,

61.2 mmol), MgCl₂(8.74 g, 91.8 mmol), and triethylamine (32.0 mL)

were dissolved in dry acetonitrile (100 mL) and stirred at room

temperature for 20 min. To the solution was added paraformaldehyde

(12.3 g, 411 mmol). The reaction mixture was stirred and heated to

reﬂux for 4 h. After cooling to room temperature, water was added,

and the mixture was acidiﬁed (pH 2.0) with diluted HCl. The mixture

was extracted with diethyl ether (50.0 mL × 2). Combined organic

layers were dried over MgSO₄and concentrated under vacuum. The

residue was puriﬁed by silica gel chromatography (ethyl acetate/

1

hexane = 1/4) to yield 10.9 g of 30 (89.1%). H NMR (400 MHz,

CDCl₃) δ 11.1 (s, 1H), 9.86 (s, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.15−

7.20 (m, 2H).

Ethyl 6-bromobenzofuran-2-carboxylate (31). A mixture of 30

(5.00 g, 25.0 mmol), K₂CO₃(10.4 g, 75.0 mmol), and bromoacetate

(2.77 mL, 25.0 mmol) in DMF (10.0 mL) was stirred at 105 °C for 1

h. After cooling to room temperature, the mixture was extracted with

ethyl acetate (50.0 mL × 2). Combined organic layers were dried over

MgSO₄and concentrated under vacuum. The residue was puriﬁed by

silica gel chromatography (ethyl acetate/hexane = 1/4) to yield 4.40 g

of 31 (65.7%). ¹H NMR (400 MHz, CDCl₃) δ 7.77 (s, 1H), 7.55 (d, J

(E)-6-Iodo-2-styrylbenzo[d]oxazole (23). The same reaction

described above for the preparation of 22 was employed to aﬀord

1

100 mg of 23 (95.1%). H NMR (400 MHz, DMSO-d₆) δ 8.16 (s,

1H), 7.81−7.85 (m, 3H), 7.71 (d, J = 8.1 Hz, 1H), 7.56 (d, J = 8.1 Hz,

1H), 7.45−7.46 (m, 3H), 7.34 (d, J = 16.5 Hz, 1H). HRMS (EI) m/z

calcd. for C₁₅H₁₀INO (M⁺) 346.9807, found 346.9813.

K

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

= 8.4 Hz, 1H), 7.49 (s, 1H), 7.44 (d, J = 8.4 Hz, 1H), 4.45 (q, J = 7.0

Hz, 2H), 1.43 (t, J = 7.2 Hz, 3H).

(E)-N,N-Dimethyl-5-(2-(6-(tributylstannyl)benzofuran-2-yl)vinyl)-

pyridin-2-amine (40). The same reaction described above for the

1

preparation of 39 was employed to aﬀord 100 mg of 40 (30.9%). H

(6-Bromobenzofuran-2-yl)methanol (32). LAH (1.87 g, 49.3

mmol) was dissolved in THF at 0 °C and 31 (4.40 g, 16.4 mmol)

of a THF solution was added, followed by stirring at 0 °C for 1 h. After

the completion of the reaction, saturated Na₂SO₄aq was added to the

resulting mixture at 0 °C and dried over Na₂SO₄. The solvent was

removed under vacuum, and the residue was puriﬁed by silica gel

chromatography (ethyl acetate/hexane = 1/1) to yield 2.10 g of 32

NMR (400 MHz, CDCl₃) δ 8.27 (d, J = 2.3 Hz, 1H), 7.67 (dd, J = 8.9,

2.5 Hz, 1H), 7.57 (s, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.18−7.25 (m,

2H), 6.78 (d, J = 16.0 Hz, 1H), 6.51−6.55 (m, 2H), 3.12 (s, 6H),

0.89−1.73 (m, 27H). MS (ESI) m/z 555.4 [MH⁺].

(E)-Tributyl(2-styrylbenzofuran-6-yl)stannane (41). The same

reaction described above for the preparation of 39 was employed to

aﬀord 65.0 mg of 41 (25.3%). ¹H NMR (400 MHz, CDCl₃) δ

7.24−7.59 (m, 9H), 7.01 (d, J = 16.0 Hz, 1H), 6.65 (s, 1H), 0.86−1.56

(m, 27H). MS (ESI) m/z 511.4 [MH⁺].

1

(56.6%). H NMR (400 MHz, CD₃OD) δ 7.63 (s, 1H), 7.43 (d, J =

8.2 Hz, 1H), 7.32 (dd, J = 8.2, 1.6 Hz, 1H), 6.68 (s, 1H), 4.65 (s, 2H).

6-Bromo-2-(bromomethyl)benzofuran (33). To a stirred solution

of 32 (2.10 g, 9.29 mmol) in diethyl ether (10.0 mL) was added

phosphorus tribromide (441 μL, 4.65 mmol) at 0 °C, followed by

stirring at room temperature for 1 h. The reaction mixture was

neutralized with saturated NaHCO₃aq and extracted with ethyl

acetate (50.0 mL × 2). Combined organic layers were dried over

MgSO₄and concentrated under vacuum. The residue was puriﬁed by

silica gel chromatography (ethyl acetate/hexane = 1/10) to yield 1.80

g of 33 (67.3%). ¹H NMR (400 MHz, CDCl₃) δ 7.66 (s, 1H), 7.40 (d,

J = 8.2 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 6.72 (s, 1H), 4.56 (s, 2H).

Diethyl ((6-bromobenzofuran-2-yl)methyl)phosphonate (34). A

mixture of 33 (1.80 g, 6.25 mmol) and triethyl phosphite (1.16 mL,

6.88 mmol) in toluene (20.0 mL) was stirred and heated to reﬂux for

24 h. After cooling to room temperature, the solvent was removed

under vacuum, and the residue was puriﬁed by silica gel

chromatography (ethyl acetate/hexane = 1/1) to yield 2.30 g of 34

(100%). ¹H NMR (400 MHz, CDCl₃) δ 7.61 (s, 1H), 7.37 (d, J = 8.4

Hz, 1H), 7.33 (dd, J = 8.4, 1.7 Hz, 1H), 6.62 (d, J = 3.8 Hz, 1H),

4.09−4.16 (m, 4H), 3.35 (d, J = 21.2 Hz, 2H), 1.29−1.36 (m, 6H).

(E)-4-(2-(6-Bromobenzofuran-2-yl)vinyl)-N,N-dimethylaniline

(35). To a solution of 34 (1.74 g, 5.03 mmol) in MeOH (30.0 mL)

was added p-dimethylaminobenzaldehyde (750 mg, 5.03 mmol) and 5

M sodium methoxide (5.00 mL, 25.0 mmol) as a base catalyst. The

reaction mixture was stirred and heated to reﬂux. After reﬂuxing for 15

h, the resulting yellow solid was collected by ﬁltration and washed with

MeOH and water. The solid was dried under vacuum to yield 475 mg

of 35 (27.7%). ¹H NMR (400 MHz, CDCl₃) δ 7.60 (s, 1H), 7.42 (d, J

= 8.7 Hz, 2H), 7.34 (d, J = 8.1 Hz, 1H), 7.29 (dd, J = 8.4, 1.7 Hz, 1H),

7.25 (d, J = 16.0 Hz, 1H), 6.76 (d, J = 16.0 Hz, 1H), 6.71 (d, J = 9.0

Hz, 2H), 6.51 (s, 1H), 3.01 (s, 6H). MS (ESI) m/z 342.1 [MH⁺].

(E)-5-(2-(6-Bromobenzofuran-2-yl)vinyl)-N,N-dimethylpyridin-2-

amine (36). The same reaction described above for the preparation of

(E)-3-(2-(6-(Tributylstannyl)benzofuran-2-yl)vinyl)pyridine (42).

The same reaction described above for the preparation of 39 was

1

employed to aﬀord 41.5 mg of 42 (21.2%). H NMR (400 MHz,

CD₃OD) δ 8.70 (d, J = 1.7 Hz, 1H), 8.41 (dd, J = 4.9, 1.7 Hz, 1H),

8.06−8.09 (m, 1H), 7.53−7.56 (m, 2H), 7.44 (dd, J = 8.1, 4.9 Hz,

1H), 7.26−7.31 (m, 3H), 6.84 (d, J = 0.9 Hz, 1H), 0.86−1.63 (m,

27H). MS (ESI) m/z 512.3 [MH⁺].

(E)-4-(2-(6-Iodobenzofuran-2-yl)vinyl)-N,N-dimethylaniline (43).

To a solution of 39 (100 mg, 0.180 mmol) in chloroform (40.0

mL) was added a solution of iodine in chloroform (3.00 mL, 50.0 mg/

mL). The mixture was stirred at room temperature for 1 h and

saturated NaHSO₃aq was added to quench the reaction. The mixture

was extracted with chloroform (50.0 mL × 2). Combined organic

layers were dried over MgSO₄and concentrated under vacuum. The

residue was puriﬁed by silica gel chromatography (ethyl acetate/

1

hexane = 1/4) to yield 18.0 mg of 43 (25.6%). H NMR (400 MHz,

CDCl₃) δ 7.79 (s, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.7 Hz,

2H), 7.22−7.24 (m, 2H), 6.75 (d, J = 16.0 Hz, 1H), 6.70 (d, J = 8.7

Hz, 2H), 6.49 (s, 1H), 2.99 (s, 6H). HRMS (EI) m/z calcd. for

C₁₈H₁₆INO (M⁺) 389.02769, found 389.02814.

(E)-5-(2-(6-Iodobenzofuran-2-yl)vinyl)-N,N-dimethylpyridin-2-

amine (44). The same reaction described above for the preparation of

1

43 was employed to aﬀord 20.0 mg of 44 (28.4%). H NMR (400

MHz, CDCl₃) δ 8.27 (d, J = 2.3 Hz, 1H), 7.79 (s, 1H), 7.66 (dd, J =

9.0, 2.3 Hz, 1H), 7.48 (dd, J = 8.1, 1.2 Hz, 1H), 7.23 (d, J = 8.1 Hz,

1H), 7.19 (d, J = 16.2 Hz, 1H), 6.73 (d, J = 16.0 Hz, 1H), 6.52−6.54

(m, 2H), 3.13 (s, 6H). HRMS (EI) m/z calcd. for C₁₇H₁₅IN₂O (M⁺)

390.02295, found 390.02270.

(E)-6-Iodo-2-styrylbenzofuran (45). The same reaction described

above for the preparation of 43 was employed to aﬀord 20.0 mg of 45

(29.5%). ¹H NMR (400 MHz, CDCl₃) δ 7.83 (s, 1H), 7.28−7.54 (m,

8H), 6.97 (d, J = 16.2 Hz, 1H), 6.63 (s, 1H). HRMS (EI) m/z calcd.

for C₁₆H₁₁IO (M⁺) 345.9855, found 345.9858.

(E)-3-(2-(6-Iodobenzofuran-2-yl)vinyl)pyridine (46). The same

reaction described above for the preparation of 43 was employed to

aﬀord 20.0 mg of 46 (74.9%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.84

(s, 1H), 8.50 (d, J = 4.9 Hz, 1H), 8.12 (d, J = 7.8 Hz, 1H), 8.01 (s,

1H), 7.58 (d, J = 8.1 Hz, 1H), 7.42−7.49 (m, 3H), 7.30 (d, J = 16.2

Hz, 1H), 7.02 (s, 1H). HRMS (EI) m/z calcd. for C₁₅H₁₀INO (M⁺)

346.9807, found 346.9810.

1

35 was employed to aﬀord 200 mg of 36 (58.5%). H NMR (400

MHz, DMSO-d₆) δ 8.29 (d, J = 2.0 Hz, 1H), 7.88 (dd, J = 9.0, 2.0 Hz,

1H), 7.81 (s, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H),

7.19 (d, J = 16.2 Hz, 1H), 7.05 (d, J = 16.2 Hz, 1H), 6.84 (s, 1H), 6.70

(d, J = 9.0 Hz, 1H), 3.07 (s, 6H). MS (ESI) m/z 343.1 [MH⁺].

(E)-6-Bromo-2-styrylbenzofuran (37). The same reaction described

above for the preparation of 35 was employed to aﬀord 198 mg of 37

(66.4%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.86 (s, 1H), 7.67 (d, J =

8.1 Hz, 2H), 7.59 (d, J = 8.1 Hz, 1H), 7.39−7.43 (m, 3H), 7.31−7.34

(m, 3H), 7.00 (s, 1H). MS (ESI) m/z 299.1 [MH⁺].

(E)-3-(2-(6-Bromobenzofuran-2-yl)vinyl)pyridine (38). The same

reaction described above for the preparation of 35 was employed to

aﬀord 117 mg of 38 (39.3%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.84

(s, 1H), 8.50 (d, J = 4.6 Hz, 1H), 8.12 (d, J = 8.1 Hz, 1H), 7.88 (s,

1H), 7.62 (d, J = 8.4 Hz, 1H), 7.42−7.49 (m, 3H), 7.31 (d, J = 16.2

Hz, 1H), 7.04 (s, 1H). MS (ESI) m/z 300.1 [MH⁺].

Methyl 6-bromobenzo[b]thiophene-2-carboxylate (47). To a

solution of 4-bromo-2-ﬂuorobenzaldehyde (808 mg, 4.00 mmol) in

DMSO (5.00 mL) were added methyl mercaptoacetate (375 μL, 4.13

mmol) and triethylamine (1.50 mL). The reaction mixture was stirred

under nitrogen atmosphere at 80 °C for 3 h. After cooling to room

temperature, the mixture was poured into water (20.0 mL) with

vigorous stirring. After 1 h of digestion, the formed solid was collected

by ﬁltration and washed with water. The solid was dried under vacuum

(E)-N,N-Dimethyl-4-(2-(6-(tributylstannyl)benzofuran-2-yl)vinyl)-

aniline (39). A mixture of 35 (171 mg, 0.500 mmol), (SnBu₃)₂(501

μL, 1.00 mmol), and (Ph₃P)₄Pd (248 mg, 0.215 mmol) in a mixed

solvent (15.0 mL, dioxane/Et₃N = 2/1) was stirred and heated to

reﬂux. After reﬂuxing for 3 h, the mixture was concentrated under

vacuum, and the residue was puriﬁed by silica gel chromatography

1

to yield 700 mg of 47 (64.8%). H NMR (400 MHz, DMSO-d₆) δ

8.40 (s, 1H), 8.22 (s, 1H), 7.98 (d, J = 8.7 Hz, 1H), 7.64 (dd, J = 8.5,

1.8 Hz, 1H), 3.90 (s, 3H).

(6-Bromobenzo[b]thiophen-2-yl)methanol (48). LAH (756 mg,

19.9 mmol) was dissolved in THF at 0 °C, and 47 (1.79 g, 6.64 mmol)

of a THF solution was added, followed by stirring at 0 °C for 3 h. After

the completion of the reaction, saturated Na₂SO₄aq was added to the

resulting mixture at 0 °C and dried over Na₂SO₄. The solvent was

removed under vacuum, and the residue was puriﬁed by silica gel

chromatography (ethyl acetate/hexane = 1/1) to yield 1.20 g of 48

1

(ethyl acetate/hexane = 1/10) to yield 100 mg of 39 (36.2%). H

NMR (400 MHz, CDCl₃) δ 7.56 (s, 1H), 7.47 (d, J = 7.3 Hz, 1H),

7.42 (d, J = 8.7 Hz, 2H), 7.23−7.27 (m, 2H), 6.81 (d, J = 16.0 Hz,

1H), 6.72 (d, J = 8.9 Hz, 2H), 6.54 (s, 1H), 3.00 (s, 6H), 0.87−1.60

(m, 27H). MS (ESI) m/z 554.4 [MH⁺].

L

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(74.7%). ¹H NMR (400 MHz, CDCl₃) δ 7.95 (s, 1H), 7.58 (d, J = 8.5

Hz, 1H), 7.44 (dd, J = 8.5, 1.8 Hz, 1H), 7.18 (s, 1H), 4.92 (d, J = 6.0

Hz, 2H), 1.89 (t, J = 6.2 Hz, 1H).

7.66 (dd, J = 9.0, 2.6 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 7.8

Hz, 1H), 7.14 (s, 1H), 7.10 (d, J = 16.0 Hz, 1H), 6.87 (d, J = 16.0 Hz,

1H), 6.53 (d, J = 8.7 Hz, 1H), 3.13 (s, 6H), 0.88−1.66 (m, 27H). MS

(ESI) m/z 571.4 [MH⁺].

6-Bromo-2-(bromomethyl)benzo[b]thiophene (49). To a stirred

solution of 48 (1.20 g, 4.96 mmol) in diethyl ether (10.0 mL) was

added phosphorus tribromide (235 μL, 2.48 mmol) at 0 °C, followed

by stirring at room temperature for 1 h. The reaction mixture was

neutralized with saturated NaHCO₃aq and extracted with ethyl

acetate (50.0 mL × 2). Combined organic layers were dried over

MgSO₄and concentrated under vacuum. The residue was puriﬁed by

silica gel chromatography (ethyl acetate/hexane = 1/10) to yield 1.51

g of 49 (100%). ¹H NMR (400 MHz, CDCl₃) δ 7.93 (s, 1H), 7.57 (d,

J = 8.5 Hz, 1H), 7.45 (dd, J = 8.5, 1.8 Hz, 1H), 7.28 (s, 1H), 4.75 (s,

2H).

(E)-Tributyl(2-styrylbenzo[b]thiophen-6-yl)stannane (57). The

same reaction described above for the preparation of 55 was employed

to aﬀord 59.0 mg of 57 (19.3%). ¹H NMR (400 MHz, CDCl₃) δ 7.86

(s, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H), 7.34−7.40

(m, 4H), 7.29 (d, J = 6.1 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H), 6.99 (d, J

= 16.0 Hz, 1H), 0.88−1.60 (m, 27H). MS (ESI) m/z 527.3 [MH⁺].

(E)-3-(2-(6-(Tributylstannyl)benzo[b]thiophen-2-yl)vinyl)pyridine

(58). The same reaction described above for the preparation of 55 was

1

employed to aﬀord 35.2 mg of 58 (27.0%). H NMR (400 MHz,

CDCl₃) δ 8.73 (s, 1H), 8.49 (d, J = 4.9 Hz, 1H), 7.87 (s, 1H), 7.81 (d,

J = 7.8 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.36−7.42 (m, 2H), 7.26−

7.30 (m, 2H), 6.94 (d, J = 16.0 Hz, 1H), 0.88−1.60 (m, 27H). MS

(ESI) m/z 528.3 [MH⁺].

Diethyl ((6-bromobenzo[b]thiophen-2-yl)methyl)phosphonate

(50). A mixture of 49 (1.51 g, 4.96 mmol) and triethyl phosphite

(4.17 mL, 24.8 mmol) in toluene (40.0 mL) was stirred and heated to

reﬂux for 44 h. After cooling to room temperature, the solvent was

removed under vacuum, and the residue was puriﬁed by silica gel

chromatography (ethyl acetate/hexane = 1/1) to yield 2.47 g of 50

(100%). ¹H NMR (400 MHz, CDCl₃) δ 7.90 (s, 1H), 7.55 (d, J = 8.5

Hz, 1H), 7.42 (dd, J = 8.5, 1.8 Hz, 1H), 7.18 (d, J = 3.2 Hz, 1H),

4.07−4.19 (m, 4H), 3.40 (d, J = 21.3 Hz, 2H), 1.28−1.39 (m, 6H).

(E)-4-(2-(6-Bromobenzo[b]thiophen-2-yl)vinyl)-N,N-dimethylani-

line (51). To a solution of 50 (2.81 g, 7.76 mmol) in MeOH (30.0

mL) were added p-dimethylaminobenzaldehyde (1.16 g, 7.76 mmol)

and 5 M sodium methoxide (7.76 mL, 38.8 mmol) as a base catalyst.

The reaction mixture was stirred and heated to reﬂux. After reﬂuxing

for 24 h, the resulting yellow solid was collected by ﬁltration and

washed with MeOH and water. The solid was dried under vacuum to

(E)-4-(2-(6-Iodobenzo[b]thiophen-2-yl)vinyl)-N,N-dimethylaniline

(59). To a solution of 55 (100 mg, 0.180 mmol) in chloroform (40.0

mL) was added a solution of iodine in chloroform (5.00 mL, 50.0 mg/

mL). The mixture was stirred at room temperature for 20 min, and

saturated NaHSO₃aq was added to quench the reaction. The mixture

was extracted with chloroform (50.0 mL × 2). Combined organic

layers were dried over MgSO₄and concentrated under vacuum. The

residue was puriﬁed by silica gel chromatography (ethyl acetate/

hexane = 1/10) to yield 20.0 mg of 59 (28.1%). ¹H NMR (400 MHz,

DMSO-d₆) δ 8.31 (s, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.54 (d, J = 8.1

Hz, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.26−7.31 (m, 2H), 6.93 (d, J =

15.9 Hz, 1H), 6.72 (d, J = 8.1 Hz, 2H), 2.95 (s, 6H). HRMS (EI) m/z

calcd. for C₁₈H₁₆INS (M⁺) 405.00484, found 405.00538.

1

(E)-5-(2-(6-Iodobenzo[b]thiophen-2-yl)vinyl)-N,N-dimethylpyri-

din-2-amine (60). The same reaction described above for the

preparation of 59 was employed to aﬀord 20.0 mg of 60 (28.1%).

¹H NMR (400 MHz, CDCl₃) δ 8.24 (d, J = 2.0 Hz, 1H), 8.07 (s, 1H),

yield 500 mg of 51 (18.0%). H NMR (400 MHz, CDCl₃) δ 7.87 (s,

1H), 7.50 (d, J = 8.7 Hz, 1H), 7.37−7.41 (m, 3H), 7.06−7.10 (m,

2H), 6.92 (d, J = 16.0 Hz, 1H), 6.71 (d, J = 9.0 Hz, 2H), 3.00 (s, 6H).

MS (ESI) m/z 358.1 [MH⁺].

7.65 (dd, J = 9.0, 2.6 Hz, 1H), 7.57 (dd, J = 8.1, 1.4 Hz, 1H), 7.38 (d, J

= 8.4 Hz, 1H), 7.09 (s, 1H), 7.05 (d, J = 16.0 Hz, 1H), 6.87 (d, J =

16.0 Hz, 1H), 6.53 (d, J = 8.7 Hz, 1H), 3.13 (s, 6H). HRMS (EI) m/z

calcd. for C₁₇H₁₅IN₂S (M⁺) 406.00010, found 405.99981.

(E)-5-(2-(6-Bromobenzo[b]thiophen-2-yl)vinyl)-N,N-dimethylpyri-

din-2-amine (52). The same reaction described above for the

preparation of 51 was employed to aﬀord 100 mg of 52 (27.9%).

¹H NMR (400 MHz, CDCl₃) δ 8.25 (d, J = 2.3 Hz, 1H), 7.88 (s, 1H),

(E)-6-Iodo-2-styrylbenzo[b]thiophene (61). The same reaction

7.66 (dd, J = 8.7, 2.3 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.40 (dd, J =

8.4, 1.7 Hz, 1H), 7.11 (s, 1H), 7.06 (d, J = 16.0 Hz, 1H), 6.87 (d, J =

16.0 Hz, 1H), 6.54 (d, J = 9.0 Hz, 1H), 3.14 (s, 6H). MS (ESI) m/z

359.1 [MH⁺].

described above for the preparation of 59 was employed to aﬀord

1

35.0 mg of 61 (86.2%). H NMR (400 MHz, CDCl₃) δ 8.10 (s, 1H),

7.59 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H), 7.42 (d, J = 8.1 Hz,

1H), 7.35−7.39 (m, 2H), 7.26−7.30 (m, 2H), 7.18 (s, 1H), 7.00 (d, J

= 16.2 Hz, 1H). HRMS (EI) m/z calcd. for C₁₆H₁₁IS (M⁺) 361.9626,

found 361.9631.

(E)-6-Bromo-2-styrylbenzo[b]thiophene (53). The same reaction

described above for the preparation of 51 was employed to aﬀord 182

1

mg of 53 (58.0%). H NMR (400 MHz, DMSO-d₆) δ 8.23 (s, 1H),

(E)-3-(2-(6-Iodobenzo[b]thiophen-2-yl)vinyl)pyridine (62). The

7.75 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 7.8 Hz, 2H), 7.60 (d, J = 16.2 Hz,

1H), 7.49−7.52 (m, 2H), 7.38−7.42 (m, 2H), 7.29−7.33 (m, 1H),

7.06 (d, J = 16.0 Hz, 1H). MS (ESI) m/z 315.1 [MH⁺].

same reaction described above for the preparation of 59 was employed

1

to aﬀord 22.6 mg of 62 (100%). H NMR (400 MHz, CDCl₃) δ 8.73

(d, J = 2.0 Hz, 1H), 8.51 (d, J = 4.6 Hz, 1H), 8.12 (s, 1H), 7.81 (d, J =

8.1 Hz, 1H), 7.62 (dd, J = 8.4, 1.7 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H),

7.34 (d, J = 16.2 Hz, 1H), 7.30 (dd, J = 7.8, 4.6 Hz, 1H), 7.24 (s, 1H),

6.96 (d, J = 16.0 Hz, 1H). HRMS (EI) m/z calcd. for C₁₅H₁₀INS (M⁺)

362.9579, found 362.9586.

(E)-3-(2-(6-Bromobenzo[b]thiophen-2-yl)vinyl)pyridine (54). The

same reaction described above for the preparation of 51 was employed

1

to aﬀord 77.0 mg of 54 (24.4%). H NMR (400 MHz, DMSO-d₆) δ

8.82 (s, 1H), 8.49 (d, J = 4.6 Hz, 1H), 8.26 (s, 1H), 8.09 (d, J = 8.1

Hz, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.74 (d, J = 16.2 Hz, 1H), 7.51−

7.53 (m, 2H), 7.42 (dd, J = 8.1, 4.9 Hz, 1H), 7.10 (d, J = 16.2 Hz, 1H).

MS (ESI) m/z 316.1 [MH⁺].

4-Iodobenzene-1,2-diamine (63). To a mixture of 4-iodo-2-

nitroaniline (2.64 g, 10.0 mmol) and concentrated HCl (2.50 mL)

in 80% EtOH (60.0 mL) was added powdered iron (2.23 g, 40.0

mmol). The reaction mixture was stirred and heated to reﬂux for 6 h.

After cooling to room temperature, the precipitate of iron oxides and

hydroxy salts was removed by ﬁltration. The solvent was removed

under vacuum, and the residue was neutralized with saturated

NaHCO₃aq and extracted with ethyl acetate (100 mL × 2).

Combined organic layers were dried over MgSO₄and concentrated

under vacuum. The residue was puriﬁed by silica gel chromatography

(E)-N,N-Dimethyl-4-(2-(6-(tributylstannyl)benzo[b]thiophen-2-

yl)vinyl)aniline (55). A mixture of 51 (179 mg, 0.500 mmol), (SnBu₃)₂

(501 μL, 1.00 mmol), and (Ph₃P)₄Pd (248 mg, 0.215 mmol) in a

mixed solvent (15.0 mL, dioxane/Et₃N = 2/1) was stirred and heated

to reﬂux. After reﬂuxing for 3 h, the mixture was concentrated under

vacuum, and the residue was puriﬁed by silica gel chromatography

1

(ethyl acetate/hexane = 1/10) to give 100 mg of 55 (35.1%). H

NMR (400 MHz, CDCl₃) δ 7.83 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H),

7.39 (d, J = 8.7 Hz, 2H), 7.36 (d, J = 7.5 Hz, 1H), 7.09−7.13 (m, 2H),

6.93 (d, J = 16.0 Hz, 1H), 6.69 (d, J = 9.0 Hz, 2H), 2.97 (s, 6H), 0.88−

1.60 (m, 27H). MS (ESI) m/z 570.4 [MH⁺].

(E)-N,N-Dimethyl-5-(2-(6-(tributylstannyl)benzo[b]thiophen-2-

yl)vinyl)pyridin-2-amine (56). The same reaction described above for

the preparation of 55 was employed to aﬀord 100 mg of 56 (35.1%).

¹H NMR (400 MHz, CDCl₃) δ 8.25 (d, J = 2.3 Hz, 1H), 7.83 (s, 1H),

1

(ethyl acetate/hexane = 1/1) to yield 1.98 g of 63 (89.1%). H NMR

(400 MHz, DMSO-d₆) δ 6.78 (d, J = 2.0 Hz, 1H), 6.62 (d, J = 8.0 Hz,

1H), 6.30 (d, J = 8.0 Hz, 1H), 4.63 (s, 2H), 4.57 (s, 2H). MS (APCI)

m/z 235 [MH⁺].

(E)-6-Iodo-2-styryl-1H-benzo[d]imidazole (64). A mixture of 63

(234 mg, 1.00 mmol), cinnamaldehyde (126 μL, 1.00 mmol), and

Na₂S₂O₅(190 mg, 1.00 mmol) dissolved in 5.00 mL of DMF was

M

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

stirred at 105 °C for 2 h. The mixture was extracted with ethyl acetate

(100 mL × 2). Combined organic layers were dried over MgSO₄and

concentrated under vacuum. The residue was puriﬁed by silica gel

chromatography (ethyl acetate/hexane = 1/1) to yield 200 mg of 64

8.13 (d, J = 8.1 Hz, 1H), 7.90 (s, 1H), 7.60 (d, J = 16.8 Hz, 1H), 7.53

(dd, J = 8.7, 1.7 Hz, 1H), 7.49 (dd, J = 7.8, 4.9 Hz, 1H), 7.35 (d, J =

8.4 Hz, 1H), 7.25 (d, J = 16.5 Hz, 1H). HRMS (EI) m/z calcd. for

C₁₄H₁₀IN₃(M⁺) 346.9920, found 346.9915.

1

(57.8%). H NMR (400 MHz, DMSO-d₆) δ 7.90 (s, 1H), 7.67−7.71

(E)-5-(2-(6-Bromo-1H-benzo[d]imidazol-2-yl)vinyl)-N,N-dimethyl-

pyridin-2-amine (72). The same reaction described above for the

(m, 3H), 7.43−7.48 (m, 3H), 7.36−7.39 (m, 2H), 7.22 (d, J = 16.5

Hz, 1H). HRMS (EI) m/z calcd. for C₁₅H₁₁IN₂(M⁺) 345.9967, found

345.9973.

1

preparation of 64 was employed to aﬀord 648 mg of 72 (77.6%). H

NMR (400 MHz, DMSO-d₆) δ 8.30 (d, J = 2.3 Hz, 1H), 7.96 (dd, J =

8.9, 2.3 Hz, 1H), 7.73 (d, J = 1.8 Hz, 1H), 7.63 (d, J = 16.5 Hz, 1H),

7.50 (d, J = 8.5 Hz, 1H), 7.34 (dd, J = 8.5, 1.8 Hz, 1H), 6.99 (d, J =

16.5 Hz, 1H), 6.78 (d, J = 9.2 Hz, 1H), 3.11 (s, 6H). MS (ESI) m/z

343.1 [MH⁺].

(E)-6-Bromo-2-(2-(pyridin-3-yl)vinyl)-1H-benzo[d]imidazole (73).

The same reaction described above for the preparation of 64 was

employed to aﬀord 176 mg of 73 (58.9%). ¹H NMR (400 MHz,

CDCl₃) δ 8.73 (d, J = 2.0 Hz, 1H), 8.50 (dd, J = 4.9, 1.7 Hz, 1H), 8.12

(d, J = 7.8 Hz, 1H), 7.69 (s, 1H), 7.59 (d, J = 16.5 Hz, 1H), 7.45−7.50

(m, 2H), 7.35 (dd, J = 8.7, 1.7 Hz, 1H), 7.24 (d, J = 16.5 Hz, 1H). MS

(ESI) m/z 300.1 [MH⁺].

tert-Butyl (E)-6-bromo-2-(2-(6-(dimethylamino)pyridin-3-yl)-

vinyl)-1H-benzo[d]imidazole-1-carboxylate (74). The same reaction

described above for the preparation of 66 was employed to aﬀord 710

mg of 74 (84.8%). ¹H NMR (400 MHz, CDCl₃) δ 8.36 (d, J = 2.3 Hz,

1H), 8.09 (d, J = 2.1 Hz, 1H), 7.90 (d, J = 15.8 Hz, 1H), 7.79 (dd, J =

9.2, 2.5 Hz, 1H), 7.67 (d, J = 15.8 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H),

7.44 (dd, J = 8.5, 2.1 Hz, 1H), 6.55 (d, J = 8.9 Hz, 1H), 3.16 (s, 6H),

1.75 (s, 9H). MS (ESI) m/z 443.2 [MH⁺].

tert-Butyl (E)-6-bromo-2-(2-(pyridin-3-yl)vinyl)-1H-benzo[d]-

imidazole-1-carboxylate (75). The same reaction described above

for the preparation of 66 was employed to aﬀord 137 mg of 75

(58.2%). ¹H NMR (400 MHz, CDCl₃) δ 8.84 (s, 1H), 8.57 (d, J = 4.6

Hz, 1H), 7.87−8.01 (m, 4H), 7.79 (d, J = 8.7 Hz, 1H), 7.43 (dd, J =

8.7, 1.7 Hz, 1H), 7.34 (dd, J = 7.8, 4.6 Hz, 1H), 1.75 (s, 9H). MS

(ESI) m/z 400.2 [MH⁺].

(E)-6-Bromo-2-styryl-1H-benzo[d]imidazole (65). The same re-

action described above for the preparation of 64 was employed to

aﬀord 200 mg of 65 (67.1%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.78

(s, 1H), 7.67−7.71 (m, 3H), 7.43−7.54 (m, 3H), 7.31−7.40 (m, 2H),

7.31 (s, 1H), 7.22 (d, J = 16.5 Hz, 1H). MS (ESI) m/z 299.1 [MH⁺].

tert-Butyl (E)-6-bromo-2-styryl-1H-benzo[d]imidazole-1-carboxy-

late (66). A mixture of 65 (200 mg, 0.670 mmol), di-tert-butyl

dicarbonate (293 mg, 1.34 mmol), DMAP (catalytic), triethylamine

(5.00 mL), and dichloromethane (10.0 mL) was stirred at room

temperature for 24 h. The reaction mixture was extracted with

chloroform (100 mL × 2). Combined organic layers were dried over

MgSO₄and concentrated under vacuum. The residue was puriﬁed by

silica gel chromatography (ethyl acetate/hexane = 1/4) to yield 200

1

mg of 66 (74.9%). H NMR (400 MHz, DMSO-d₆) δ 7.65−8.08 (m,

6H), 7.41−7.55 (m, 4H), 1.70 (s, 9H). MS (ESI) m/z 399.2 [MH⁺].

tert-Butyl (E)-2-styryl-6-(tributylstannyl)-1H-benzo[d]imidazole-

1-carboxylate (67). A mixture of 66 (200 mg, 0.500 mmol),

(SnBu₃)₂(583 μL, 1.00 mmol), and (Ph₃P)₄Pd (250 mg, 0.220

mmol) in a mixed solvent (10.0 mL, dioxane/Et₃N = 1/1) was stirred

and heated to reﬂux. After reﬂuxing for 2 h, the mixture was

concentrated under vacuum, and the residue was puriﬁed by silica gel

chromatography (ethyl acetate/hexane = 1/10) to yield 41.0 mg of 67

1

(13.4%). H NMR (400 MHz, CDCl₃) δ 7.92−8.03 (m, 3H), 7.63−

7.76 (m, 3H), 7.32−7.43 (m, 4H), 1.76 (s, 9H), 0.88−1.63 (m, 27H).

MS (ESI) m/z 611.4 [MH⁺].

(E)-3-(6-(Dimethylamino)pyridin-3-yl)acrylaldehyde (68). To a

solution of 6-(dimethylamino)nicotinaldehyde (600 mg, 4.00 mmol),

(1,3-dioxolan-2-ylmethyl)triphenylphosphonium bromide (3.43 g,

8.00 mmol), and 18-crown-6 (catalytic) in THF (25.0 mL) was

added NaH (384 mg, 16.0 mmol) in one portion. After stirring at

room temperature for 2 h, the same amount of NaH was added, and

the stirring was continued overnight. The reaction mixture was

quenched with water and extracted with diethyl ether (50.0 mL × 3).

Combined organic layers were removed under vacuum. The residue

was dissolved in a mixture of THF (50.0 mL) and 10% HCl (20.0 mL)

and stirred at room temperature for 6 h. The reaction mixture was

alkalized with 10% NaOH aq and extracted with dichloromethane

(100 mL × 2). Combined organic layers were dried over MgSO₄and

concentrated under vacuum. The residue was puriﬁed by silica gel

chromatography (ethyl acetate/hexane = 2/1) to yield 1.22 g of 68

(86.6%). ¹H NMR (400 MHz, CDCl₃) δ 9.61 (d, J = 7.8 Hz, 1H), 8.30

(d, J = 2.3 Hz, 1H), 7.68 (dd, J = 9.2, 2.5 Hz, 1H), 7.37 (d, J = 15.6

Hz, 1H), 6.50−6.57 (m, 2H), 3.18 (s, 6H). MS (ESI) m/z 177.2

[MH⁺].

tert-Butyl (E)-2-(2-(6-(dimethylamino)pyridin-3-yl)vinyl)-6-(tribu-

tylstannyl)-1H-benzo[d]imidazole-1-carboxylate (76). The same

reaction described above for the preparation of 67 was employed to

1

aﬀord 30.0 mg of 76 (18.4%). H NMR (400 MHz, CDCl₃) δ 8.16−

8.36 (m, 1H), 7.68−8.00 (m, 3H), 7.31−7.41 (m, 2H), 6.15−6.56 (m,

1H), 4.91−5.32 (m, 1H), 2.90−3.15 (m, 6H), 1.68−1.75 (m, 9H),

0.84−1.65 (m, 27H). MS (ESI) m/z 655.5 [MH⁺].

tert-Butyl (E)-2-(2-(pyridin-3-yl)vinyl)-6-(tributylstannyl)-1H-

benzo[d]imidazole-1-carboxylate (77). The same reaction described

above for the preparation of 67 was employed to aﬀord 31.1 mg of 77

(25.9%). ¹H NMR (400 MHz, CDCl₃) δ 8.84 (d, J = 1.7 Hz, 1H), 8.56

(d, J = 3.8 Hz, 1H), 8.05 (d, J = 16.0 Hz, 1H), 7.88−7.97 (m, 4H),

7.42 (d, J = 7.8 Hz, 1H), 7.33 (dd, J = 8.1, 4.9 Hz, 1H), 1.75 (s, 9H),

0.87−1.60 (m, 27H). MS (ESI) m/z 612.4 [MH⁺].

Immunohistochemical Staining Using AD Brain Sections.

Post-mortem brain tissues from an autopsy-conﬁrmed case of AD (a

76-year-old male) were obtained from Graduate School of Medicine,

Kyoto University. Six-micrometer-thick serial sections of paraﬃn-

embedded blocks were used for staining. The sections were subjected

to two 15 min incubations in xylene, two 1 min incubations in 100%

EtOH, one 1 min incubation in 90% EtOH, one 1 min incubation in

80% EtOH, and one 1 min incubation in 70% EtOH to completely

deparaﬃnize them, followed by two 2.5 min washes in H₂O. They

were then autoclaved for 15 min in 0.01 M citric acid buﬀer (pH 6.8)

and incubated with 90% formic acid for 5 min to activate the antigen.

After two 5 min incubations in PBS-Tween 20, the sections were

incubated at room temperature with an antiphosphorylated tau (AT8,

Thermo Scientiﬁc) or Aβ₁₋₄₂(BC05, Wako) primary antibody for 1 h.

After three 5 min incubations in PBS-Tween 20, they were incubated

with biotinylated goat antimouse IgG (Histoﬁne Simple Stain Mouse

MAX-PO (MULTI), Nichirei Biosciences Inc.) at room temperature

for 30 min. After three 3 min incubations in PBS-Tween 20 and one 5

min incubation in TBS, the sections were incubated with DAB as a

chromogen for 1 min. After being washed with H₂O, the sections were

observed under a microscope (BIOREVO BZ-9000, Keyence Corp.).

(E)-3-(Pyridin-3-yl)acrylaldehyde (69). The same reaction de-

scribed above for the preparation of 68 was employed to aﬀord 565

mg of 69 (85.0%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.71 (d, J = 7.5

Hz, 1H), 8.91 (s, 1H), 8.64 (d, J = 4.6 Hz, 1H), 8.22 (d, J = 8.1 Hz,

1H), 7.80 (d, J = 15.9 Hz, 1H), 7.51 (dd, J = 8.1, 4.9 Hz, 1H), 7.02

(dd, J = 16.0, 7.5 Hz, 1H).

(E)-5-(2-(6-Iodo-1H-benzo[d]imidazol-2-yl)vinyl)-N,N-dimethyl-

pyridin-2-amine (70). The same reaction described above for the

1

preparation of 64 was employed to aﬀord 100 mg of 70 (25.6%). H

NMR (400 MHz, DMSO-d₆) δ 8.32 (s, 1H), 8.02−8.05 (m, 2H), 7.80

(d, J = 16.2 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 8.4 Hz,

1H), 7.03 (d, J = 16.2 Hz, 1H), 6.88 (d, J = 9.3 Hz, 1H), 3.15 (s, 6H).

HRMS (EI) m/z calcd. for C₁₆H₁₅IN₄(M⁺) 390.0342, found

390.0335.

(E)-6-Iodo-2-(2-(pyridin-3-yl)vinyl)-1H-benzo[d]imidazole (71).

The same reaction described above for the preparation of 64 was

1

employed to aﬀord 73.6 mg of 71 (21.2%). H NMR (400 MHz,

CD₃OD) δ 8.74 (d, J = 2.0 Hz, 1H), 8.50 (dd, J = 4.6, 1.5 Hz, 1H),

N

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Radiolabeling. The radioiodinated forms of ligands were prepared

from the corresponding tributyltin precursors by iododestannylation

according to the conventional method^41,42(see the detailed method in

Supporting Information). See the solvent used in radiolabeling, HPLC

conditions used for the puriﬁcation of each ligand, and the retention

time of each ligand in Table S1.

In Vitro Autoradiography. Post-mortem brain tissues from an

autopsy-conﬁrmed case of AD (a 76-year-old male) and a healthy

control (a 73-year-old male) were obtained from Graduate School of

Medicine, Kyoto University and BioChain Institute Inc., respectively.

The presence and location of tau and Aβ deposits in the sections were

conﬁrmed with immunohistochemical staining using an antiphosphoy-

lated tau antibody (AT8) and Aβ₁₋₄₂antibody (BC05), respectively.

Deparaﬃnization was carried out according to the procedure described

above. The sections were incubated with radioiodinated ligands (370

kBq/mL in 50% EtOH) for 2 h at room temperature. They were then

dipped in 50% EtOH for 2 h and washed with H₂O for 30 s. After

drying, the ¹²⁵I-labeled sections were exposed to a BAS imaging plate

(Fuji Film) overnight. Autoradiographic images were obtained using a

BAS5000 scanner system (Fuji Film).

Measurement of log D_7.4Values. The experimental determi-

nation of partition coeﬃcients of each radioiodinated ligand was

performed in 1-octanol and 0.01 M PBS (−) at a pH of 7.4 according

to the conventional method⁴²(see the detailed method in Supporting

Information).

In Vivo Biodistribution in Normal Mice. The experiments with

animals were conducted in accordance with our institutional guidelines

and approved by Kyoto University. A saline solution (100 μL) of each

radioiodinated ligand (19.6−29.4 kBq) containing EtOH (10.0 μL)

and Tween 80 (0.100 μL) was injected intravenously directly into the

tails of ddY mice (5-week-old male). The mice were sacriﬁced at

various time points postinjection. The organs of interest were removed

and weighed, and the radioactivity was measured with an automatic γ

counter (Wizard 1470, PerkinElmer).

Technology Policy (CSTP), Grant-in-Aid for Scientiﬁc

Research on Innovative Areas (Brain Protein Aging and

Dementia Control (15H01555) from MEXT, and JSPS

Research Fellowships for Young Scientists.

ABBREVIATIONS USED

■

AD, Alzheimer’s disease; Aβ, amyloid β peptides; SP, senile

plaques; NFT, neuroﬁbrillary tangles; PET, positron emission

tomography; SPECT, single photon emission computed

tomography; CSF, cerebrospinal ﬂuid; NMR, nuclear magnetic

resonance; MS (ESI or APCI), mass spectrometry (electrospray

ionization or atmospheric pressure chemical ionization);

HRMS (EI), high-resolution mass spectrometry (electron

impact); Na₂S₂O₅, sodium metabisulﬁte; Boc, tert-butoxycar-

bonyl; HPLC, high-performance liquid chromatography; TFA,

triﬂuoroacetic acid; NaI, sodium iodide; TMS, tetramethylsi-

lane; J, coupling constant (in NMR spectrometry); MgSO₄,

magnesium sulfate; CuI, copper(I) iodide; Na₂S·9H₂O, sodium

sulﬁde nonahydrate; DMF, N,N-dimethylformamide; HCl,

hydrochloric acid; NaHCO₃, sodium hydrogen carbonate;

NaOH, sodium hydroxide; DMSO, dimethyl sulfoxide;

(SnBu₃)₂, bis(tributyltin); (Ph₃P)₄Pd, tetrakis-

(triphenylphosphine)palladium(0); Et₃N, triethylamine;

NaHSO₃, sodium hydrogen sulﬁte; K₂CO₃, potassium carbo-

nate; MgCl₂, magnesium chloride; LAH, lithium aluminum

hydride; TBS, tris-buﬀered saline; THF, tetrahydrofuran;

DMAP, N,N-dimethyl-4-aminopyridine; NaH, sodium hydride;

DAB, 3,3′-diaminobenzidine; H₂O₂, hydrogen peroxide;

AcOH, acetic acid; DBDMH, 1,3-dibromo-5,5-dimethylhydan-

toin; PPA, polyphosphoric acid; (HCHO)_n, paraformaldehyde;

MeCN, acetonitrile; PBr₃, phosphorus tribromide; Et₂O,

diethyl ether; P(OEt)₃, triethyl phosphite; NaOMe, sodium

methoxide; PBS, phosphate buﬀered saline; (CH₃CO)₂O,

acetic anhydride

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.5b00440.

REFERENCES

■

(1) Brookmeyer, R.; Johnson, E.; Ziegler-Graham, K.; Arrighi, H. M.

Forecasting the global burden of Alzheimer’s disease. Alzheimer's

Dementia 2007, 3, 186−191.

(2) Felsenstein, K. M.; Candelario, K. M.; Steindler, D. A.; Borchelt,

D. R. Regenerative medicine in Alzheimer’s disease. Transl Res. 2014,

163, 432−438.

Summary of the method and solvent used in radio-

labeling, HPLC condition used for the puriﬁcation of

each ligand, the retention time of each ligand in the

HPLC analysis, biodistribution of radioactivity after

injection of [¹²⁵I]64 in normal mice, in vitro

ﬂuorescent-staining image of compound 3 in the AD

brain section, in vitro autoradiography of [¹²⁵I]64 in the

healthy control brain section, quantitative analysis of

autoradiography of each ligand, evaluation of in vitro and

(3) Jack, C. R., Jr.; Knopman, D. S.; Jagust, W. J.; Shaw, L. M.; Aisen,

P. S.; Weiner, M. W.; Petersen, R. C.; Trojanowski, J. Q. Hypothetical

model of dynamic biomarkers of the Alzheimer’s pathological cascade.

Lancet Neurol. 2010, 9, 119−128.

(4) Mathis, C. A.; Mason, N. S.; Lopresti, B. J.; Klunk, W. E.

Development of positron emission tomography β-amyloid plaque

imaging agents. Semin. Nucl. Med. 2012, 42, 423−432.

(5) Ono, M.; Wilson, A.; Nobrega, J.; Westaway, D.; Verhoeff, P.;

Zhuang, Z. P.; Kung, M. P.; Kung, H. F. ¹¹C-labeled stilbene

derivatives as Aβ-aggregate-specific PET imaging agents for

Alzheimer’s disease. Nucl. Med. Biol. 2003, 30, 565−571.

1

in vivo stability of [¹²⁵I]64, H NMR spectra ﬁles, and

detailed methods for radiolabeling and measurements of

log D_7.4values (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone +81-75-753-4608. Fax: +81-75-753-4568. E-mail:

ono@pharm.kyoto-u.ac.jp.

■

(6) Furukawa, K.; Okamura, N.; Tashiro, M.; Waragai, M.;

Furumoto, S.; Iwata, R.; Yanai, K.; Kudo, Y.; Arai, H. Amyloid PET

in mild cognitive impairment and Alzheimer’s disease with BF-227:

comparison to FDG-PET. J. Neurol. 2010, 257, 721−727.

(7) Vandenberghe, R.; Van Laere, K.; Ivanoiu, A.; Salmon, E.; Bastin,

C.; Triau, E.; Hasselbalch, S.; Law, I.; Andersen, A.; Korner, A.;

Minthon, L.; Garraux, G.; Nelissen, N.; Bormans, G.; Buckley, C.;

Owenius, R.; Thurfjell, L.; Farrar, G.; Brooks, D. J. ¹⁸F-flutemetamol

amyloid imaging in Alzheimer disease and mild cognitive impairment:

a phase 2 trial. Ann. Neurol. 2010, 68, 319−329.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This research was supported by a grant from the Japan Society

for the Promotion of Science (JSPS) through the “Funding

Program for Next Generation World-Leading Researchers

(NEXT Program),” initiated by the Council for Science and

(8) Kung, H. F.; Choi, S. R.; Qu, W.; Zhang, W.; Skovronsky, D. ¹⁸F

stilbenes and styrylpyridines for PET imaging of Aβ plaques in

O

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Alzheimer’s disease: a miniperspective. J. Med. Chem. 2010, 53, 933−

941.

(23) Delacourte, A.; David, J. P.; Sergeant, N.; Buee, L.; Wattez, A.;

Vermersch, P.; Ghozali, F.; Fallet-Bianco, C.; Pasquier, F.; Lebert, F.;

Petit, H.; Di Menza, C. The biochemical pathway of neurofibrillary

degeneration in aging and Alzheimer’s disease. Neurology 1999, 52,

1158−1165.

(24) Gomez-Isla, T.; Hollister, R.; West, H.; Mui, S.; Growdon, J. H.;

Petersen, R. C.; Parisi, J. E.; Hyman, B. T. Neuronal loss correlates

with but exceeds neurofibrillary tangles in Alzheimer’s disease. Ann.

Neurol. 1997, 41, 17−24.

(25) Nelson, P. T.; Alafuzoff, I.; Bigio, E. H.; Bouras, C.; Braak, H.;

Cairns, N. J.; Castellani, R. J.; Crain, B. J.; Davies, P.; Del Tredici, K.;

Duyckaerts, C.; Frosch, M. P.; Haroutunian, V.; Hof, P. R.; Hulette, C.

M.; Hyman, B. T.; Iwatsubo, T.; Jellinger, K. A.; Jicha, G. A.; Kovari,

E.; Kukull, W. A.; Leverenz, J. B.; Love, S.; Mackenzie, I. R.; Mann, D.

M.; Masliah, E.; McKee, A. C.; Montine, T. J.; Morris, J. C.; Schneider,

J. A.; Sonnen, J. A.; Thal, D. R.; Trojanowski, J. Q.; Troncoso, J. C.;

Wisniewski, T.; Woltjer, R. L.; Beach, T. G. Correlation of Alzheimer

disease neuropathologic changes with cognitive status: a review of the

literature. J. Neuropathol. Exp. Neurol. 2012, 71, 362−381.

(26) Whitwell, J. L.; Josephs, K. A.; Murray, M. E.; Kantarci, K.;

Przybelski, S. A.; Weigand, S. D.; Vemuri, P.; Senjem, M. L.; Parisi, J.

E.; Knopman, D. S.; Boeve, B. F.; Petersen, R. C.; Dickson, D. W.;

Jack, C. R., Jr. MRI correlates of neurofibrillary tangle pathology at

autopsy: a voxel-based morphometry study. Neurology 2008, 71, 743−

749.

(9) Clark, C. M.; Schneider, J. A.; Bedell, B. J.; Beach, T. G.; Bilker,

W. B.; Mintun, M. A.; Pontecorvo, M. J.; Hefti, F.; Carpenter, A. P.;

Flitter, M. L.; Krautkramer, M. J.; Kung, H. F.; Coleman, R. E.;

Doraiswamy, P. M.; Fleisher, A. S.; Sabbagh, M. N.; Sadowsky, C. H.;

Reiman, E. P.; Zehntner, S. P.; Skovronsky, D. M.; Group, A. A. S. Use

of florbetapir-PET for imaging β-amyloid pathology. JAMA 2011, 305,

275−283.

(10) Kung, M. P.; Hou, C.; Zhuang, Z. P.; Zhang, B.; Skovronsky, D.;

Trojanowski, J. Q.; Lee, V. M.; Kung, H. F. IMPY: an improved

thioflavin-T derivative for in vivo labeling of β-amyloid plaques. Brain

Res. 2002, 956, 202−210.

(11) Rowe, C. C.; Pejoska, S.; Mulligan, R. S.; Jones, G.; Chan, J. G.;

Svensson, S.; Cselenyi, Z.; Masters, C. L.; Villemagne, V. L. Head-to-

head comparison of ¹¹C-PiB and ¹⁸F-AZD4694 (NAV4694) for β-

amyloid imaging in aging and dementia. J. Nucl. Med. 2013, 54, 880−

886.

(12) Yousefi, B. H.; Manook, A.; Grimmer, T.; Arzberger, T.; von

Reutern, B.; Henriksen, G.; Drzezga, A.; Forster, S.; Schwaiger, M.;

Wester, H. J. Characterization and first human investigation of FIBT, a

novel fluorinated Aβ plaque neuroimaging PET radioligand. ACS

Chem. Neurosci. 2015, 6, 428−437.

(13) Jack, C. R., Jr.; Lowe, V. J.; Weigand, S. D.; Wiste, H. J.; Senjem,

M. L.; Knopman, D. S.; Shiung, M. M.; Gunter, J. L.; Boeve, B. F.;

Kemp, B. J.; Weiner, M.; Petersen, R. C. Alzheimer’s Disease

Neuroimaging, I. Serial PIB and MRI in normal, mild cognitive

impairment and Alzheimer’s disease: implications for sequence of

pathological events in Alzheimer’s disease. Brain 2009, 132, 1355−

1365.

(14) Mintun, M. A.; Larossa, G. N.; Sheline, Y. I.; Dence, C. S.; Lee,

S. Y.; Mach, R. H.; Klunk, W. E.; Mathis, C. A.; DeKosky, S. T.;

Morris, J. C. [¹¹C]PIB in a nondemented population: potential

antecedent marker of Alzheimer disease. Neurology 2006, 67, 446−

452.

(15) Aizenstein, H. J.; Nebes, R. D.; Saxton, J. A.; Price, J. C.; Mathis,

C. A.; Tsopelas, N. D.; Ziolko, S. K.; James, J. A.; Snitz, B. E.; Houck,

P. R.; Bi, W.; Cohen, A. D.; Lopresti, B. J.; DeKosky, S. T.; Halligan, E.

M.; Klunk, W. E. Frequent amyloid deposition without significant

cognitive impairment among the elderly. Arch. Neurol. 2008, 65,

1509−1517.

(16) Citron, M. Alzheimer’s disease: strategies for disease

modification. Nat. Rev. Drug Discovery 2010, 9, 387−398.

(17) Mullard, A. Sting of Alzheimer’s failures offset by upcoming

prevention trials. Nat. Rev. Drug Discovery 2012, 11, 657−660.

(18) Gilman, S.; Koller, M.; Black, R. S.; Jenkins, L.; Griffith, S. G.;

Fox, N. C.; Eisner, L.; Kirby, L.; Rovira, M. B.; Forette, F.; Orgogozo,

J. M.; Team, A. N. S. Clinical effects of Aβ immunization (AN1792) in

patients with AD in an interrupted trial. Neurology 2005, 64, 1553−

1562.

(27) Okamura, N.; Furumoto, S.; Fodero-Tavoletti, M. T.; Mulligan,

R. S.; Harada, R.; Yates, P.; Pejoska, S.; Kudo, Y.; Masters, C. L.; Yanai,

K.; Rowe, C. C.; Villemagne, V. L. Non-invasive assessment of

Alzheimer’s disease neurofibrillary pathology using ¹⁸F-THK5105

PET. Brain 2014, 137, 1762−1771.

(28) Okamura, N.; Furumoto, S.; Harada, R.; Tago, T.; Yoshikawa,

T.; Fodero-Tavoletti, M.; Mulligan, R. S.; Villemagne, V. L.; Akatsu,

H.; Yamamoto, T.; Arai, H.; Iwata, R.; Yanai, K.; Kudo, Y. Novel ¹⁸F-

labeled arylquinoline derivatives for noninvasive imaging of tau

pathology in Alzheimer disease. J. Nucl. Med. 2013, 54, 1420−1427.

(29) Harada, R.; Okamura, N.; Furumoto, S.; Furukawa, K.; Ishiki,

A.; Tomita, N.; Hiraoka, K.; Watanuki, S.; Shidahara, M.; Miyake, M.;

Ishikawa, Y.; Matsuda, R.; Inami, A.; Yoshikawa, T.; Tago, T.; Funaki,

Y.; Iwata, R.; Tashiro, M.; Yanai, K.; Arai, H.; Kudo, Y. [¹⁸F]THK-

5117 PET for assessing neurofibrillary pathology in Alzheimer’s

disease. Eur. J. Nucl. Med. Mol. Imaging 2015, 42, 1052−1061.

(30) Xia, C. F.; Arteaga, J.; Chen, G.; Gangadharmath, U.; Gomez, L.

F.; Kasi, D.; Lam, C.; Liang, Q.; Liu, C.; Mocharla, V. P.; Mu, F.;

Sinha, A.; Su, H.; Szardenings, A. K.; Walsh, J. C.; Wang, E.; Yu, C.;

Zhang, W.; Zhao, T.; Kolb, H. C. [¹⁸F]T807, a novel tau positron

emission tomography imaging agent for Alzheimer’s disease.

Alzheimer's Dementia 2013, 9, 666−676.

(31) Chien, D. T.; Bahri, S.; Szardenings, A. K.; Walsh, J. C.; Mu, F.;

Su, M. Y.; Shankle, W. R.; Elizarov, A.; Kolb, H. C. Early clinical PET

imaging results with the novel PHF-tau radioligand [F-18]-T807. J.

Alzheimers Dis 2013, 34, 457−468.

(32) Chien, D. T.; Szardenings, A. K.; Bahri, S.; Walsh, J. C.; Mu, F.;

Xia, C.; Shankle, W. R.; Lerner, A. J.; Su, M. Y.; Elizarov, A.; Kolb, H.

C. Early clinical PET imaging results with the novel PHF-tau

radioligand [F18]-T808. J. Alzheimers Dis 2014, 38, 171−184.

(33) Zhang, W.; Arteaga, J.; Cashion, D. K.; Chen, G.;

Gangadharmath, U.; Gomez, L. F.; Kasi, D.; Lam, C.; Liang, Q.; Liu,

C.; Mocharla, V. P.; Mu, F.; Sinha, A.; Szardenings, A. K.; Wang, E.;

Walsh, J. C.; Xia, C.; Yu, C.; Zhao, T.; Kolb, H. C. A highly selective

and specific PET tracer for imaging of tau pathologies. J. Alzheimers

Dis 2012, 31, 601−612.

(19) Holmes, C.; Boche, D.; Wilkinson, D.; Yadegarfar, G.; Hopkins,

V.; Bayer, A.; Jones, R. W.; Bullock, R.; Love, S.; Neal, J. W.; Zotova,

E.; Nicoll, J. A. Long-term effects of Aβ₄₂immunisation in Alzheimer’s

disease: follow-up of a randomised, placebo-controlled phase I trial.

Lancet 2008, 372, 216−223.

(20) Jack, C. R., Jr.; Knopman, D. S.; Jagust, W. J.; Petersen, R. C.;

Weiner, M. W.; Aisen, P. S.; Shaw, L. M.; Vemuri, P.; Wiste, H. J.;

Weigand, S. D.; Lesnick, T. G.; Pankratz, V. S.; Donohue, M. C.;

Trojanowski, J. Q. Tracking pathophysiological processes in

Alzheimer’s disease: an updated hypothetical model of dynamic

biomarkers. Lancet Neurol. 2013, 12, 207−216.

(21) Shaw, L. M.; Vanderstichele, H.; Knapik-Czajka, M.; Clark, C.

M.; Aisen, P. S.; Petersen, R. C.; Blennow, K.; Soares, H.; Simon, A.;

Lewczuk, P.; Dean, R.; Siemers, E.; Potter, W.; Lee, V. M.;

Trojanowski, J. Q. Alzheimer’s Disease Neuroimaging, I. Cerebrospinal

fluid biomarker signature in Alzheimer’s disease neuroimaging

initiative subjects. Ann. Neurol. 2009, 65, 403−413.

(34) Maruyama, M.; Shimada, H.; Suhara, T.; Shinotoh, H.; Ji, B.;

Maeda, J.; Zhang, M. R.; Trojanowski, J. Q.; Lee, V. M.; Ono, M.;

Masamoto, K.; Takano, H.; Sahara, N.; Iwata, N.; Okamura, N.;

Furumoto, S.; Kudo, Y.; Chang, Q.; Saido, T. C.; Takashima, A.;

Lewis, J.; Jang, M. K.; Aoki, I.; Ito, H.; Higuchi, M. Imaging of tau

pathology in a tauopathy mouse model and in Alzheimer patients

compared to normal controls. Neuron 2013, 79, 1094−1108.

(22) Braak, H.; Braak, E. Neuropathological stageing of Alzheimer-

related changes. Acta Neuropathol. 1991, 82, 239−259.

P

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(35) Hashimoto, H.; Kawamura, K.; Igarashi, N.; Takei, M.;

Fujishiro, T.; Aihara, Y.; Shiomi, S.; Muto, M.; Ito, T.; Furutsuka,

K.; Yamasaki, T.; Yui, J.; Xie, L.; Ono, M.; Hatori, A.; Nemoto, K.;

Suhara, T.; Higuchi, M.; Zhang, M. R. Radiosynthesis, photo-

isomerization, biodistribution, and metabolite analysis of ¹¹C-PBB3

as a clinically useful PET probe for imaging of tau pathology. J. Nucl.

Med. 2014, 55, 1532−1538.

(36) Shao, X.; Carpenter, G. M.; Desmond, T. J.; Sherman, P.;

Quesada, C. A.; Fawaz, M.; Brooks, A. F.; Kilbourn, M. R.; Albin, R. L.;

Frey, K. A.; Scott, P. J. Evaluation of [¹¹C]N-Methyl Lansoprazole as a

Radiopharmaceutical for PET Imaging of Tau Neurofibrillary Tangles.

ACS Med. Chem. Lett. 2012, 3, 936−941.

(37) Fawaz, M. V.; Brooks, A. F.; Rodnick, M. E.; Carpenter, G. M.;

Shao, X.; Desmond, T. J.; Sherman, P.; Quesada, C. A.; Hockley, B. G.;

Kilbourn, M. R.; Albin, R. L.; Frey, K. A.; Scott, P. J. High affinity

radiopharmaceuticals based upon lansoprazole for PET imaging of

aggregated tau in Alzheimer’s disease and progressive supranuclear

palsy: synthesis, preclinical evaluation, and lead selection. ACS Chem.

Neurosci. 2014, 5, 718−730.

M.; Maruyama, M.; Arai, H.; Yanai, K.; Sawada, T.; Kudo, Y. Quinoline

and benzimidazole derivatives: candidate probes for in vivo imaging of

tau pathology in Alzheimer’s disease. J. Neurosci. 2005, 25, 10857−

10862.

(50) Harada, R.; Okamura, N.; Furumoto, S.; Yoshikawa, T.; Arai, H.;

Yanai, K.; Kudo, Y. Use of a benzimidazole derivative BF-188 in

fluorescence multispectral imaging for selective visualization of tau

protein fibrils in the Alzheimer’s disease brain. Mol. Imaging Biol. 2014,

16, 19−27.

(51) Pike, V. W. PET radiotracers: crossing the blood-brain barrier

and surviving metabolism. Trends Pharmacol. Sci. 2009, 30, 431−440.

(52) Snellman, A.; Rokka, J.; Lopez-Picon, F. R.; Eskola, O.; Wilson,

I.; Farrar, G.; Scheinin, M.; Solin, O.; Rinne, J. O.; Haaparanta-Solin,

M. Pharmacokinetics of [¹⁸F]flutemetamol in wild-type rodents and its

binding to beta amyloid deposits in a mouse model of Alzheimer’s

disease. Eur. J. Nucl. Med. Mol. Imaging 2012, 39, 1784−1795.

(38) Riss, P.; Brichard, L.; Ferrari, V.; Williamson, D.; Fryer, T.;

Hong, Y.; Baron, J.; Aigbirhio, F. Radiosynthesis and characterization

of astemizole derivatives as lead compounds toward PET imaging of τ-

pathology. MedChemComm 2013, 4, 852−855.

(39) Ono, M.; Hayashi, S.; Matsumura, K.; Kimura, H.; Okamoto, Y.;

Ihara, M.; Takahashi, R.; Mori, H.; Saji, H. Rhodanine and

thiohydantoin derivatives for detecting tau pathology in Alzheimer’s

brains. ACS Chem. Neurosci. 2011, 2, 269−275.

(40) Watanabe, H.; Ono, M.; Kimura, H.; Matsumura, K.;

Yoshimura, M.; Okamoto, Y.; Ihara, M.; Takahashi, R.; Saji, H.

Synthesis and biological evaluation of novel oxindole derivatives for

imaging neurofibrillary tangles in Alzheimer’s disease. Bioorg. Med.

Chem. Lett. 2012, 22, 5700−5703.

(41) Matsumura, K.; Ono, M.; Hayashi, H.; Kimura, H.; Okamoto,

Y.; Ihara, M.; Takahashi, K.; Mori, H.; Saji, H. Phenyldiazenyl

benzothiazole derivatives as probes for in vivo imaging of neuro-

fibrillary tangles in Alzheimer’s disease brains. MedChemComm 2011,

2, 596−600.

(42) Matsumura, K.; Ono, M.; Yoshimura, M.; Kimura, H.;

Watanabe, H.; Okamoto, Y.; Ihara, M.; Takahashi, R.; Saji, H.

Synthesis and biological evaluation of novel styryl benzimidazole

derivatives as probes for imaging of neurofibrillary tangles in

Alzheimer’s disease. Bioorg. Med. Chem. 2013, 21, 3356−3362.

(43) Watanabe, H.; Ono, M.; Iikuni, S.; Kimura, H.; Okamoto, Y.;

Ihara, M.; Saji, H. Synthesis and biological evaluation of ¹²³I-labeled

pyridyl benzoxazole derivatives: novel β-amyloid imaging probes for

single-photon emission computed tomography. RSC Adv. 2015, 5,

1009−1015.

(44) Ono, M.; Cheng, Y.; Kimura, H.; Watanabe, H.; Matsumura, K.;

Yoshimura, M.; Iikuni, S.; Okamoto, Y.; Ihara, M.; Takahashi, R.; Saji,

H. Development of novel ¹²³I-labeled pyridyl benzofuran derivatives

for SPECT imaging of β-amyloid plaques in Alzheimer’s disease. PLoS

One 2013, 8, e74104.

(45) Mishra, A.; Thangamani, A.; Chatterjee, S.; Chipem, F. A.;

Krishnamoorthy, G. Photoisomerization of trans-2-[4′-

(dimethylamino)styryl]benzothiazole. Photochem. Photobiol. 2013, 89,

247−252.

(46) Lewis, F. D.; Yoon, B. A.; Arai, T.; Iwasaki, T.; Tokumatsu, K.

Molecular structure and photochemistry of (E)- and (Z)-2-(2-(2-

pyridyl)ethenyl)indole. A case of hydrogen bond dependent one-way

photoisomerization. J. Am. Chem. Soc. 1995, 117, 3029−3036.

(47) Rojo, L. E.; Alzate-Morales, J.; Saavedra, I. N.; Davies, P.;

Maccioni, R. B. Selective interaction of lansoprazole and astemizole

with tau polymers: potential new clinical use in diagnosis of

Alzheimer’s disease. J. Alzheimers Dis 2010, 19, 573−589.

(48) Honson, N. S.; Johnson, R. L.; Huang, W.; Inglese, J.; Austin, C.

P.; Kuret, J. Differentiating Alzheimer disease-associated aggregates

with small molecules. Neurobiol. Dis. 2007, 28, 251−260.

(49) Okamura, N.; Suemoto, T.; Furumoto, S.; Suzuki, M.;

Shimadzu, H.; Akatsu, H.; Yamamoto, T.; Fujiwara, H.; Nemoto,

Q

DOI: 10.1021/acs.jmedchem.5b00440

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Structure-Activity Relationship Study of Heterocyclic Phenylethenyl and Pyridinylethenyl Derivatives as Tau-Imaging Agents That Selectively Detect Neurofibrillary Tangles in Alzheimers Disease Brains

DOI: 10.1021/acs.jmedchem.5b00440

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.5b00440

Full text of DOI:10.1021/acs.jmedchem.5b00440

Products guided by the article

R&D Labs maybe for 1089681-53-7

Relevant to this article

Hot Product