Design and synthesis of a monocyclic derivative as a selective ACC1 inhibitor by chemical modification of biphenyl ACC1/2 dual inhibitors

Article abstract of DOI:10.1016/j.bmc.2021.116056

A structure–activity relationship (SAR) study towards novel ACC1-selective inhibitors was carried out by modifying the molecular length of the linker in biaryl derivative 1 g, an ACC1/2 dual inhibitor. Ultimately, this leads us to discover novel phenoxybenzyloxy derivative 1i as a potent ACC1-selective inhibitor. Further chemical modification of this scaffold to improve cellular potency as well as physicochemical and pharmacokinetic (PK) properties produced N-2-(pyridin-2-ylethyl)acetamide derivative 1n, which showed highly potent ACC1-selective inhibition as well as sufficient PK profile for further in vivo evaluations. Oral administration of 1n significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of 100 mg/kg. Accordingly, our novel series of potent ACC1-selective inhibitors represents a set of useful orally-available research tools, as well as potential therapeutic agents for cancer and fatty acid-related diseases.

Full text of DOI:10.1016/j.bmc.2021.116056

Bioorg. Med. Chem. 35 (2021) 116056
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of a monocyclic derivative as a selective ACC1
inhibitor by chemical modification of biphenyl ACC1/2 dual inhibitors
Ryo Mizojiri*, Daisuke Tomita1, Masako Sasaki, Yoshihiko Satoh2, Yukiko Yamamoto,
Hiroyuki Sumi3, Hironobu Maezaki4
Research, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
A structure–activity relationship (SAR) study towards novel ACC1-selective inhibitors was carried out by
modifying the molecular length of the linker in biaryl derivative 1 g, an ACC1/2 dual inhibitor. Ultimately, this
leads us to discover novel phenoxybenzyloxy derivative 1i as a potent ACC1-selective inhibitor. Further chemical
modification of this scaffold to improve cellular potency as well as physicochemical and pharmacokinetic (PK)
properties produced N-2-(pyridin-2-ylethyl)acetamide derivative 1n, which showed highly potent ACC1-
selective inhibition as well as sufficient PK profile for further in vivo evaluations. Oral administration of 1n
significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of 100 mg/kg.
Accordingly, our novel series of potent ACC1-selective inhibitors represents a set of useful orally-available
research tools, as well as potential therapeutic agents for cancer and fatty acid-related diseases.
Acetyl-CoA carboxylase (ACC) 1 inhibitor
Selectivity
14C acetate uptake inhibition
Bioavailability
Malonyl-CoA
1. Introduction
treatment of fatty acid metabolism disorders, whereas selective ACC1
inhibitors have been rarely reported. Recently, we reported novel ACC1-
Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in de
novo lipogenesis and plays an important role in the regulation of fatty
acid metabolism.1–5 Therefore, ACC inhibition provides a promising
selective inhibitors as in vivo probe molecules and demonstrated anti-
tumor activity for cancer therapeutics. 34–36
Among those reports, Haque et al. described the discovery of potent
biphenyl-based ACC inhibitors.27 In the report, it is interesting to note
that compound 1b possessing 4-propoxy tail unit showed potent dual
ACC1/2 inhibitory activity, on the other hand, compound 1c possessing
4-methoxyethyl tail unit demonstrated excellent ACC1-selective inhi-
bition. These results indicate that oxygen atom in the tail unit would be
effective to adjust selectivity for these molecules. Furthermore, the po-
sitional relationship of the oxygen in the tail unit to the acetamide
moiety in the head part of the molecule, an essential moiety for potent
ACCs inhibitory activity, could potentially give rise to a new series of
selective ACC1 inhibitors. Therefore, we hypothesized that the insertion
of an appropriate linker between biphenyl moieties of 1 would lead us to
6
7
therapeutic potential in type 2 diabetes (T2DM), obesity, nonalco-
8–10
11–15
holic fatty liver disease (NAFLD),
cancer
and immunology.
16,17
Two ACC isoforms have been identified in mammals, ACC1 and
ACC2. Malonyl-CoA which is produced by ACC1 is an intermediate of de
novo fatty acid synthesis, which acts as a substrate of fatty acid synthase
(FAS) for acyl chain elongation. On the other hand, ACC2, a mito-
chondrial membrane-associated enzyme, generates malonyl-CoA pri-
marily to inhibit carnitine palmitoyltransferase 1 (CPT-1) and regulate
fatty acid transport into the mitochondria to enhance fatty acid oxida-
tion (FAO). A number of mammalian ACC inhibitors have been reported
as dual ACC1/2 inhibitors18–30 or selective ACC2 inhibitors28–33 for the
Abbreviations: ATP, adenosine triphosphate; BSA, bovine serum albumin; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DIAD, diisopropyl azodicarboxylate; IPE,
diisopropyl ether; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; DPPA, diphenylphosphoryl azide; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HOBt, 1-hydroxybenzotriazole; MsCl, methanesulfonyl chloride; PBS, phosphate buffered saline;
THF, tetrahydrofuran.
* Corresponding author.
E-mail address: ryo.mizojiri@takeda.com (R. Mizojiri).
1
2
Otsuka Pharmaceutical Co., Ltd., Minato-ku, Tokyo 108-8242, Japan.
Chordia Therapeutics Inc., Fujisawa-shi, Kanagawa 251-8585, Japan.
3
Daiichi Sankyo Co., Ltd., Shinagawa-ku, Tokyo 140-0005, Japan.
4
Axcelead Drug Discovery Partners Inc., Fujisawa-shi, Kanagawa 251-8585, Japan.
https://doi.org/10.1016/j.bmc.2021.116056
Received 8 December 2020; Received in revised form 28 January 2021; Accepted 30 January 2021
Available online 14 February 2021
0968-0896/© 2021 The Authors.
Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
discover a novel ACC1-selective inhibitor as shown in Figure 1.
Herein, we report the discovery of a novel series of selective ACC1
inhibitors using a different approach than we previously had re-
ported,34 as well as the in vivo activity in xenograft tumors.
hydrolysis followed by amide formation produced 29. Subsequent re-
action with MeMgBr, followed by reductive amination and lastly acet-
ylation gave compounds 1n and 1o.
Preparation of compound 1p is described in Scheme 7. Copper-
catalyzed coupling reaction of 24 with phenol 10 gave diphenyl ether
derivative 30. Substitution reaction of 3,6-dibromopyridazine (31) with
30 yielded bromopyridazine derivative 32. Introduction of vinylether by
Pd-catalyzed coupling reaction followed by hydrolysis under acidic
condition introduced a ketone moiety. After reduction of ketone moiety
in 33 into alcohol, azide derivative was synthesized via meth-
ansulfonylation followed by azidation with sodium azide. Finally,
compound 1p was synthesized via Staudinger reduction of azide group
followed by acetylation.
1.1. Chemistry
Preparation of biaryl compounds 1a–g begins with the copper-
catalyzed coupling reaction of 1-(4′-bromo-[1,1′-biphenyl]-4-yl)ethan-
1-one (2) with corresponding phenols (3) to give phenyl ether de-
rivatives (4). After conversion of ketones 4 into amines 6 by reduction to
alcohol, then azidation with DPPA, followed by reduction of azide,
compounds 1a–e were synthesized by acetylation of the amino group in
amines 6. Lastly, alkylation of phenol moiety of 1e yielded 1f and 1 g
(Scheme 1).
Preparation of compound 1q is illustrated in Scheme 8. Substitution
reaction of 1-(5-chloropyrazin-2-yl)ethan-1-one (36) with 30 under
microwave irradiation gave 37. Conversion of ketone moiety in 37 into
acetamide via reductive amination followed by acetylation yielded
compound 1q.
Preparation of compound 1 h is displayed in Scheme 2. Coupling
reaction of 4-iodophenol (7) and 1-(4-fluorophenyl)ethan-1-one (8)
followed by copper-catalyzed coupling reaction with phenol 10 gave
1,4-diphenoxyphenyl derivative 11. Conversion of carbonyl group in 11
into amino group by reductive amination followed by acetylation pro-
duced compound 1 h.
2. Results and discussion
1. Activity and selectivity for ACC1 inhibition of N-(1-phenylethyl)
Preparation of compounds 1i, 1j and 1 l is illustrated in Scheme 3.
Acetylation of 4-(1-aminoethyl)phenol (12) gave acetamide 13.
Coupling reaction of phenol 10 with corresponding aryl and heteroaryl
halides 14 produced biaryl ethers 15 which was subjected to reduction
with LiAlH4 or NaBH4 to give benzyl alcohol derivatives 16. Mitsunobu
reaction of 16a and 16c with 13 yielded compounds 1i and 1 l,
respectively. After methanesulfonylation of 16b, compound 1j was
synthesized by the coupling reaction with 13.
acetamide derivatives
Compounds prepared in this study were evaluated for their inhibi-
tory activity against recombinant human ACC1 and ACC2 expressed in
SF-9 cells. To measure the inhibition of de novo lipid synthesis in cells of
our ACC1 inhibitors, we established a 14C-acetate uptake assay in HCT-
116 colon cancer cells. To begin our investigation, we assessed biphenyl
ACC1 inhibitors 1a–c, reported by Haque et al.,27 using our enzyme
assay condition (Table 1). Under our enzyme assay condition, reported
compounds 1a–c showed moderate ACC1 inhibitory potency (around
Preparation of compound 1 k is shown in Scheme 4. Mitsunobu re-
action of (5-bromopyridin-2-yl)methanol (17) with 1-(4-hydrox-
yphenyl)ethan-1-one (18) gave phenoxy derivative 19. Copper-
catalyzed coupling reaction of 19 with phenol 10 yielded 20. Reduc-
tion of ketone 20 followed by azidation with DPPA produced azide 21.
Finally, hydrogenation of the azide followed by acetylation afforded 1 k.
Preparation of compound 1 m is explained in Scheme 5. Acetamide
derivative 25 was synthesized via reduction of 2,6-difluoro-4-iodoben-
zaldehyde (23) with NaBH4 followed by Mitsunobu reaction with
phenol 13. Copper-catalyzed coupling reaction of 25 with phenol 10
gave compound 1 m.
10 μM as IC50 value). Therefore, we tried to identify more potent ACCs
inhibitor for our lead compound. During our chemical modification,
replacement of tail unit at C4-position into C3-position dramatically
improved ACC1 inhibitory potency (1d: hACC1 IC50 = 130 nM, hACC2
IC50 = 98 nM) compared to compound 1c. After minor modification of
tail unit at C3-position, compound 1 g possessing 3-cyclopropylmethoxy
group showed potent ACC1/2 dual inhibition (hACC1 IC50 = 60 nM,
hACC2 IC50 = 150 nM). Accordingly, we initiated our synthetic strategy
to insert appropriate linker between biphenyl moieties of this
compound.
Preparation of compounds 1n and 1o is shown in Scheme 6. Sub-
stitution reaction of 24 with fluoropyridines 26 gave benzyl ether de-
rivative 27. Copper-catalyzed coupling reaction of 27 with phenol 10
yielded 28. Conversion of ester group in 28 into Weinreb amide via
At the beginning of the investigation for a novel ACC1-selective in-
hibitor, our lead compound 1 g showed potent ACC1 inhibitory potency
ACC1/2 dual inhibitor
ACC1 selective inhibitor
Head part
O
O
N
Tail unit
N
H
H
O
O
O
O
1b
1c
hACC1 IC50 = 17 nM
hACC2 IC50 = 14 nM
hACC1 IC50 = 89 nM
hACC2 IC50 >30000 nM
O
N
H
Linker
O
R
O
1
Fig. 1. Synthetic strategy for the generation of novel ACC1-selective inhibitorsa. aInhibitory activities refer the data in the literature.
2

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
Scheme 1. Reagents and conditions: a) N, N-dimethylglycine hydrochloride, CuI, Cs2CO3, DMF, 140 ◦C, overnight, 17–52%; b) i) NaBH4, THF, MeOH, 0 ◦C, 1 h; ii)
DPPA, DBU, toluene, rt, 2 h, 46–100%; c) for 1a–1c and 1e: H2 (1 atm), Pd/C, THF, rt, 2 h; d) for 1d: Ph3P, THF, water, 60 ◦C, 1 h, 62%; e) Ac2O, pyridine, rt, 30 min,
5–98%; f) R’Br, K2CO3, DMF, 80 ◦C, 2 h, 40–49%.
Scheme 2. Reagents and conditions: a) K2CO3, DMF, 50 ◦C, overnight, 69%; b) 3-(cyclopropylmethoxy)phenol (10), N,N-Dimethylglycine hydrochloride, CuI,
Cs2CO3, DMF, 90 ◦C, overnight, 56%; c) i) NH4OAc, NaBH3CN, MeOH, 60 ◦C, overnight; ii) Ac2O, Et3N, THF, rt, 3 h, 68%.
Scheme 3. Reagents and conditions: a) Ac2O, THF, rt, 2 h, 61%; b) for 15a and 15c: 10, Cs2CO3, DMF, 60–100 ◦C, 3 h to overnight, 60%; c) for 15b: 10, K2CO3,
DMF, 60 ◦C, 2 h, 53%; d) for 16a: LiAlH4, THF, 0 ◦C, 30 min, 98%; e) for 16b and 16c: NaBH4, MeOH, 0 ◦C, 2 h, 30%; f) for 1i and 1 l: 13, tributylphosphine, 1,1′-
(azodicarbonyl)-dipiperidine, toluene, rt, overnight, 30–39%, g) for 1j: MsCl, Et3N, THF, 0 ◦C, 4 h; ii) 13, K2CO3, DMF, 50 ◦C, overnight, 15%.
(ACC1 IC50 = 60 nM), however, its selectivity over ACC2 was not
enough (ca. 2.5 fold) for our research of ACC1-selective inhibitors. To
adjust the positional relationship of acetamide moiety in the head part
and ether group in the tail unit, insertion of oxygen atom between
biphenyl moiety was attempted and compound 1 h possessing 1,4-
quinone moiety in the linker section was produced as a potent ACC1/
2 dual inhibitor (hACC1 IC50 = 39 nM, hACC2 IC50 = 20 nM). This result
indicated that the position of both the head and tail units may not be
optimal for ACC1-selective inhibition. Therefore, we continued our
investigation for an alternative linker. Interestingly enough, this led us
to identify a novel 4-phenoxybenzyloxy derivative 1i, which showed
potent ACC1-selective inhibition with excellent selectivity over ACC2
(hACC1 IC50 = 220 nM, hACC2 IC50 > 10000 nM). Accordingly, we
found that the positional relationship between the acetamide moiety
and the ether group of compound 1i was essential for highly potent
ACC1-selective inhibition (Fig. 2).
Next, we conducted modification of our novel 4-phenoxybenzyloxy
derivative as a selective ACC1 inhibitor to identify in vivo probe mole-
cule (Table 2). Compound 1i showed potent ACC1-selective inhibition,
however its cellular potency of Acetate uptake inhibition in HCT-116
cells was not sufficient for further in vitro and in vivo evaluations. In
order to improve the cellular potency, we first investigated chemical
modification of the middle aromatic ring (Ring A, highlighted in yellow)
to improve ACC1 inhibitory activity. The replacement of the phenyl ring
for a pyridine ring decreased ACC1 inhibitory potency as well as cellular
activity (1j: hACC1 IC50 = 1200 nM, Acetate uptake IC50 > 1000 nM; 1
k: hACC1 IC50 = 790 nM, Acetate uptake IC50 = 900 nM) compared to
compound 1i (hACC1 IC50 = 220 nM, Acetate uptake IC50 420 nM).
Further investigation by introducing heteroaryl ring instead of phenyl
moiety was not effective in enhancing ACC1 inhibitory potency.
3

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
Scheme 4. Reagents and conditions: a) DIAD, Ph3P, THF, rt, overnight, 88%; b) 10, picolinic acid, CuI, K3PO4, DMSO, 90 ◦C, 4 h, 55%; c) i) NaBH4, EtOH, THF, rt, 4
h; ii) DPPA, DBU, toluene, rt, 2 h, 88%; d) Ph3P, water , THF, 60 ◦C, 1 h, 79%; e) Ac2O, pyridine, rt, 30 min, 76%.
Scheme 5. Reagents and conditions: a) NaBH4, EtOH, 0 ◦C, 1 h, 98%; b) 13, DIAD, Ph3P, THF, 12 ◦C, 4 h, 76%; c) 10, picolinic acid, CuI, K3PO4, DMSO, 90 ◦C,
overnight, 38%.
Scheme 6. Reagents and conditions: a) 24, NaH, DMF, 30 ◦C, 2 h, 36–53%; b) 10, picolinic acid, CuI, K3PO4, DMSO, 90 ◦C, 12–24 h, 63%; c) i) LiOH⋅H2O, THF,
MeOH, water, 26–32 ◦C, 3–12 h; ii) N.O-dimethlyhydroxylamine hydrochloride, EDCI, HOBt, Et3N, DMF, 30 ◦C, 3 h, 83%; d) i) MeMgBr, THF, 0 ◦C, 1 h; ii) NH4OAc,
NaBH3CN, MeOH, reflux, 12 h; iii) Ac2O, MeOH, 30 ◦C, 1–2 h, 9–45%.
Scheme 7. Reagents and conditions: a) 10, picolinic acid, CuI, K3PO4, DMSO, 90 ◦C, 15 h, 54%; b) 3,6-dibromopyridazine (31), NaH, THF, 18 ◦C, 4 h, 75%; c) i)
tributyl(1-ethoxyvinyl)tin, Pd(Ph3P)2Cl2, DMF, 80 ◦C, 15 h; ii) aqueous HCl, acetone, 20 ◦C, 1 h, 57%; d) NaBH4, MeOH, 20 ◦C, 0.5 h, 93%; e) i) MsCl, Et3N, CH2Cl2,
20 ◦C, 1 h; ii) NaN3, DMF, 60 ◦C, 2 h, 77%; f) i) Ph3P, THF, water, reflux, 15 h; ii) Ac2O, MeOH, 20 ◦C, 1 h, 52%.
Therefore, we then investigated the substituent on Ring A as a tight SAR
space. According to our continuous research, only small lipophilic sub-
stituent (e.g. fluorine atom) was acceptable for ACC1 inhibition. In
compound 1 l, the introduction of fluorine atom at C2-position of phenyl
ring showed potent ACC1 inhibitory activity with excellent selectivity
over ACC2 and improved cellular potency (hACC1 IC50 = 69 nM, hACC2
IC50 > 10000 nM, Acetate uptake IC50 = 63 nM). Furthermore, substi-
tution of fluorine atoms at C2 and C6-position on the middle aromatic
4

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
Scheme 8. Reagents and conditions: a) 30, 1-(5-chloropyrazin-2-yl)ethan-1-one (36), NaH, THF, 135 ◦C, 1 h, MW, 24%; b) i) NH4OAc, NaBH3CN, MeOH, 65 ◦C,
overnight; ii) Ac2O, THF, rt, 1 h, 53%.
3. In vivo evaluation of an ACC1-selective inhibitor
Table 1
Physicochemical and PK profiles for compounds 1 m and 1n are
Initial SAR studies of biphenyl derivatives a
displayed in Table 4. Both compounds showed excellent ACC1-selective
inhibition as well as potent cellular activity. On the other hand, they still
had insufficient solubility due to high lipophilicity. Further investigation
Compound
R
IC50 (nM)
ACC1
for their ADMET profiles revealed that both compounds demonstrated
sufficient properties such as permeability, metabolic stability in liver
microsomes. In terms of CYP inhibition potential, they showed accept-
able properties for CYP2C8 and CYP2C9, which were challenges with
bicyclic ACC1-selective inhibitors.34 Furthermore, they possessed good
bioavailability in mouse cassette PK study. In comparison between the
two compounds, 1n showed improved solubility under acidic conditions
and a better PK profile than 1 m, hence as a promising probe molecule
for further in vivo evaluations.
ACC2
1a
1b
1c
1d
4-(CH2)2CH3
4-O(CH2)2CH3
4-(CH2)2OCH3
3-(CH2)2OCH3
38%b
11%b
50%b
51%b
49%b
6%b
130
98
(92–191)
120
(53–181)
210
1f
3-O(CH2)3CH3
(88–165)
60
(129–337)
150
1 g
3-OCH2cPr
(35–101)
(111–212)
Based on the promising in vitro and in vivo profile of 1n, we selected
this compound for in vivo PD study to evaluate the potential of an ACC1
inhibitor in xenograft tumor models. In this PD study, malonyl-CoA
concentration in tumors was measured as a direct PD marker. As a
result, compound 1n showed potent malonyl-CoA suppression at 2 h
after single oral administration in HCT-116 xenograft mice at 100 mg/
kg, and its reduction by compound 1n was sustained up to 16 h after the
administration (Fig. 3).
a
IC50 is presented as the mean of duplicate experiments along with 95%
confidence intervals (95%CI) in parentheses. bInhibitory potency was shown as a
% inhibition at 10 μM.
ring led us to identify highly potent ACC1-selective inhibitor 1 m which
also exhibited improved cellular potency (hACC1 IC50 = 15 nM, hACC2
IC50 > 10000 nM, Acetate uptake IC50 = 9.7 nM).Table 3.
According to our investigation of Ring A, 4-phenoxy-2,6-difluoro-
benzyl derivative 1 m was identified as a highly potent ACC1-selective
4. Conclusions
inhibitor. Further physicochemical profiling revealed that
1 m
possessed low solubility due to its high lipophilicity (cLog P = 6.22,
Solubility: <0.21 mg/mL). Therefore, we next conducted modification
of phenyl ring in the head part (Ring B) to reduce its lipophilicity.
Replacement of the phenyl ring into a pyridine moiety reduced lip-
ophilicity as expected (1n and 1o: cLog P = 5.23). Among these, com-
pound 1n showed highly potent ACC1-selective inhibition (hACC1 IC50
= 17 nM, hACC2 IC50 > 10000 nM, Acetate uptake IC50 = 24 nM)
comparable to 1 m (hACC1 IC50 = 15 nM, hACC2 IC50 > 10000 nM,
Acetate uptake IC50 = 9.7 nM). Further introduction of nitrogen atom to
reduce lipophilicity gave pyridazine derivative 1p (cLog P = 4.26) and
pyrazine derivative 1q (cLog P = 4.40) which showed acceptable ACC1
inhibitory potency, however both compounds showed decreased cellular
activity compared to compound 1n in return for the reduction of lip-
ophilicity. Accordingly, compound 1 m and 1n were identified as a
highly potent ACC1-selective inhibitors possessing excellent cellular
activity.
We have investigated a new approach to discover a novel series of 4-
phenoxybenzyloxy derivatives as chemical probes of selective ACC1
inhibitors from biaryl ACC1/2 dual inhibitors by the modification of the
linker section. In the beginning of our research, we found compound 1i
showed potent ACC1 inhibitory activity with excellent selectivity over
ACC2. After chemical modification of the middle section (Ring A) of the
molecule, compound 1 m showed highly potent ACC1-selective inhibi-
tion as well as excellent cellular activity (Acetate uptake: IC50 = 9.7 nM),
however this compound possessed low solubility due to high lip-
ophilicity. Further optimization of the head part (Ring B) produced N-2-
(pyridin-2-ylethyl)acetamide derivative 1n, which showed highly
potent ACC1-selective inhibition as well as sufficient PK profile for
further in vivo evaluations. Based on the promising PK profile and re-
sults from in vivo PD study, 1n is considered to be useful in vivo probe
molecule for basic pharmacology research into ACC1 function.
ACC1/2 dual inhibitor
ACC1/2 dual inhibitor
ACC1 selective inhibitor
O
O
O
N
N
O
O
H
H
N
O
H
O
O
O
O
O
1g
1h
1i
hACC1 IC50 = 60 nM
hACC2 IC50 = 150 nM
hACC1 IC50 = 39 nM
hACC2 IC50 = 20 nM
hACC1 IC50 = 220 nM
hACC2 IC50 >10000 nM
Fig. 2. Initial SAR studies of the linker part for ACC1 inhibitors.
5

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
(MS) were acquired using a Shimadzu UFLC/MS (Prominence UFLC high
pressure gradient system/LCMS-2020) operating in an electron spray
ionization mode (ESI + ). The column used was an L-column 2 ODS (3.0
Table 2
a
Optimization for Ring
A
of 4-phenoxybenzyloxy derivatives
× 50 mm I.D., 3 μ
m, CERI, Japan) with a temperature of 40 ◦C and a flow
rate of 1.2 or 1.5 mL/min. Condition 1: Mobile phases A and B under an
acidic condition were 0.05% TFA in water and 0.05% TFA in CH3CN,
respectively. The ratio of mobile phase B was increased linearly from 5%
to 90% over 0.9 min, 90% over the next 1.1 min. The ratio of mobile
phase B was increased linearly from 5% to 90% over 0.9 min, 90% over
the next 1.1 min. The purities of all compounds tested in biological
systems were assessed as being > 95% using elemental analysis or LCMS.
Reagents and solvents were obtained from commercial sources and used
without further purification. Reaction progress was determined by thin
layer chromatography (TLC) analysis on Merck Kieselgel 60 F254 plates
or Fuji Silysia NH plates. Chromatographic purification was carried out
on silica gel columns ((Inject column and Universal column, YAMAZEN
Co.) or on Purif-Pack (Si or NH, Shoko Scientific Co., Ltd.)). All
commercially available solvents and reagents were used without further
purification. Starting materials (2, 3, 7, 8, 10, 12, 14, 17, 18, 23, 26, 31
and 36) were purchased from commercial companies, and used as such.
Yields were not optimized.
Compound
IC50 (nM)
ACC1
ACC2
Acetateb
1i
220
>10000
420
(109–428)
(223–797)
1j
790
>10000
>10000
>10000
900
(342–1819)
(398–2037)
1 k
1 l
1200
>1000
(750–1764)
69
63
(37–128)
(35–114)
1 m
15
>10000
9.7
(9.0–27)
(7.3–13)
a
IC50 is presented as the mean of duplicate experiments along with 95%
confidence intervals (95%CI) in parentheses.
5.1.1. General procedure for the preparation of compound 1a–c, e
1-[4′-(4-propylphenoxy)[1,1′-biphenyl]-4-yl]ethan-1-one (4a):
A mixture of 4-propylphenol (2a) (0.52 g, 3.8 mmol), 1-(4′-bromo[1,1′-
biphenyl]-4-yl)ethan-1-one (3) (1 g, 3.6 mmol), N,N-dimethylglycine
hydrochloride (0.051 g, 0.36 mmol), CuI (0.012 mL, 0.36 mmol) and
Cs2CO3 (1.8 g, 5.5 mmol) in DMF (10 mL) was stirred at 140 ◦C over-
night. After cooling to room temperature, the mixture was extracted
with AcOEt and water. The organic layer was washed with sat. aq. NaCl,
dried over MgSO4 and pass through a pad of NH silica gel (eluted with
AcOEt). After concentration, the residue was crystallized from EtOH to
afford 4a (0.28 g, 24%) as a beige solid.
b
Acetate uptake inhibition in HCT-116 cells.
5. Experimental section
5.1. General
Proton nuclear magnetic resonance (1H NMR) spectra were recorded
on Bruker AVANCE-300 (300 MHz) and Bruker AVANCE-400 (400 MHz)
instruments in CDCl3, CD3OD or DMSO‑d6 solution. Chemical shifts are
given in parts per million (ppm) with tetramethylsilane as an internal
standard. Abbreviations are used as follows: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, dd = doublet of doublets, dt =
doublet of triplets, brs = broad singlet. Coupling constants (J values) are
given in hertz (Hz). Melting points (mp) were determined on an Opti-
Melt MPA100 melting point apparatus and were uncorrected. Elemental
analyses were carried out by Sumika Chemical Analysis Service, Ltd. and
were within 0.4% of the theoretical values. Low-resolution mass spectra
1H NMR (300 MHz, DMSO‑d6) δ 0.91 (3H, t, J = 7.3 Hz), 1.47–1.68
(2H, m), 2.53–2.65 (5H, m), 6.92–7.12 (4H, m), 7.24 (2H, d, J = 8.5 Hz),
7.69–7.90 (4H, m), 8.02 (2H, d, J = 8.5 Hz). LCMS m/z calcd for
C
23H22O2: 330.16, found 331.1 [M + H] + .
1-[4′-(4-Propoxyphenoxy)[1,1′-biphenyl]-4-yl]ethan-1-one
(4b): Product was prepared according to the general procedure from
0.58 g of 4-propoxyphenol (3b) and obtained (0.65 g, 52%) as a white
solid.
Table 3
Optimization for Ring B of 4-phenoxy-2,6-difluorobenzyloxy derivatives.a
Compound
IC50 (nM)
ACC1
cLogP
Solubilityc
(mg/mL)
ACC2
Acetateb
1m
1n
1o
1p
1q
15
>10000
9.7
6.22
5.23
5.23
4.26
4.40
<0.21
<0.23
1.5
(9.0–27)
(7.3–13)
17
>10000
>10000
>10000
>10000
24
(10–28)
(18–30)
38
70
(24–59)
(41–120)
31
110
2.3
(20–49)
(88–131)
89
180
0.46
(49–162)
(99–310)
a
IC50 is presented as the mean of duplicate experiments along with 95% confidence intervals (95%CI) in parentheses.
b
c
Acetate uptake inhibition in HCT-116 cells.
Solubility in pH 6.8.
6

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
one (4e): Product was prepared according to the general procedure from
1 g of 3-(benzyloxy)phenol (3e) and obtained (0.32 g, 17%) as a white
solid.
Table 4
Physicochemical and PK profiles of benzyloxy derivatives.a
Compound
IC50 (nM)
ACC1
Acetate uptake IC50 (nM)b
1 m
1n
1H NMR (300 MHz, DMSO‑d6) δ 2.61 (3H, s), 5.11 (2H, s), 6.60–6.68
(1H, m), 6.74 (1H, t, J = 2.3 Hz), 6.80–6.89 (1H, m), 7.12 (2H, d, J = 8.7
Hz), 7.28–7.46 (6H, m), 7.80 (4H, dd, J = 11.9, 8.6 Hz), 8.03 (2H, d, J =
8.5 Hz). LCMS m/z calcd for C27H22O3: 394.16, found 395.1 [M + H] + .
4-(1-Azidoethyl)-4′-(4-propylphenoxy)-1,1′-biphenyl (5a): To an
ice cold stirred solution of 4a (284 mg, 0.86 mmol) in THF (5 mL) and
MeOH (2 mL) was added NaBH4 (33 mg, 0.86 mmol). After stirring at 0
◦C for 1 h, the mixture was extracted with AcOEt and 1 N HCl. The
organic layer was washed with sat. aq. NaCl, dried over MgSO4 and
concentrated under reduced pressure to afford the alcohol. A mixture of
the alcohol, DPPA (473 mg, 1.7 mmol) and DBU (0.26 mL, 1.7 mmol) in
toluene (5 mL) was stirred at room temperature for 2 h. The mixture was
extracted with toluene and water. The organic layer was washed with
sat. aq. NaCl, dried over MgSO4 and concentrated under reduced pres-
sure. The residue was purified by column chromatography (silica gel,
eluent: 5/95 to 25/75 AcOEt/hexane) to afford 5a (190 mg, 62%).
1H NMR (300 MHz, DMSO‑d6) δ 0.90 (3H, t, J = 7.3 Hz), 1.49 (3H, d,
J = 6.8 Hz), 1.54–1.67 (2H, m), 2.53–2.61 (2H, m), 4.80–4.96 (1H, m),
6.93–7.10 (4H, m), 7.23 (2H, d, J = 8.5 Hz), 7.46 (2H, d, J = 8.2 Hz),
7.61–7.74 (4H, m).
15
>10000
17
>10000
ACC2
(9.0–27)
(10–28)
9.7
24
(7.3–13)
6.22
(18–30)
5.23
0.96
cLogP
LogD
5.01
4.82
Solubilityc (mg/mL)
pH1.2
<0.23
<0.21
<0.23
pH6.8
PAMPA (nm/sec)
pH7.4
111
346
683
662
pH5.0
Metabolic Stability (
μ
L/min/mg)
30
36
12
17
human
mouse
CYP inhibition (%)
2C8
31.4
1584.1
ꢀ 22.7
31.1
32.0
5346.0
ꢀ 13.8
70.5
2C9
Mouse Cassette PKd
AUCe (ng•h/mL)
Ff (%)
Reagents and conditions: a) N, N-dimethylglycine hydrochloride, CuI, Cs2CO3,
DMF, 140 ◦C, overnight, 17–52%; b) i) NaBH4, THF, MeOH, 0 ◦C, 1 h; ii) DPPA,
DBU, toluene, rt, 2 h, 46–100%; c) for 1a–1c and 1e: H2 (1 atm), Pd/C, THF, rt, 2
h; d) for 1d: Ph3P, THF, water, 60 ◦C, 1 h, 62%; e) Ac2O, pyridine, rt, 30 min,
5–98%; f) R’Br, K2CO3, DMF, 80 ◦C, 2
a
IC50 is presented as the mean of duplicate experiments along with 95%
confidence intervals (95%CI) in parentheses. bAcetate uptake inhibition in HCT-
116 cells. cSolubility in pH 6.8. dMale ICR mice (n = 3). Dose: i.v. at 0.1 mg/kg;
p.o. at 1 mg/kg. eArea under the curve from 0 to 8 h. fBioavailability.
4-(1-Azidoethyl)-4′-(4-propoxyphenoxy)-1,1′-biphenyl
(5b):
Product was prepared according to the general procedure from 651 mg
of 4b and obtained (321 mg, 46%).
1H NMR (300 MHz, DMSO‑d6) δ 0.99 (3H, t, J = 7.4 Hz), 1.49 (3H, d,
J = 6.8 Hz), 1.63–1.84 (2H, m, J = 7.3 Hz), 3.92 (2H, t, J = 6.5 Hz),
4.72–5.02 (1H, m), 6.86–7.11 (6H, m), 7.45 (2H, d, J = 8.2 Hz), 7.65
(4H, d, J = 8.4 Hz).
0.50
#: P<0.05 by Dunnet t-test
0.45
0.40
0.35
#
0.30
0.25
4-(1-Azidoethyl)-4′-[4-(2-methoxyethyl)phenoxy]-1,1′-
biphenyl (5c): Product was prepared according to the general proced-
ure from 292 mg of 4c and obtained (200 mg, 64%).
#
0.20
0.15
0.10
0.05
0.00
1H NMR (300 MHz, DMSO‑d6) δ 1.49 (3H, d, J = 6.8 Hz), 2.81 (2H, t,
J = 6.8 Hz), 3.25 (3H, s), 3.54 (2H, t, J = 6.8 Hz), 4.88 (1H, d, J = 6.7
Hz), 6.94–7.10 (4H, m), 7.27 (2H, d, J = 8.3 Hz), 7.46 (2H, d, J = 8.0
Hz), 7.67 (4H, dd, J = 8.3, 4.7 Hz).
Vehicle 100 mg/kg Vehicle 100 mg/kg
Compound
1n
Compound
1n
4-(1-Azidoethyl)-4′-[3-(2-methoxyethyl)phenoxy]-1,1′-
biphenyl (5d): Product was prepared according to the general proced-
ure from 0.9 g of 4d and obtained (0.97 g, 100%).
2h
16h
1H NMR (300 MHz, DMSO‑d6) δ 1.49 (3H, d, J = 6.9 Hz), 2.81 (2H, t,
J = 6.8 Hz), 3.23 (3H, s), 3.46–3.60 (2H, m), 4.89 (1H, q, J = 6.8 Hz),
6.84–6.94 (1H, m), 6.98 (1H, d, J = 2.1 Hz), 7.02–7.11 (3H, m),
7.28–7.32 (1H, m), 7.47–7.51 (4H, m), 7.69–7.73 (2H, m).
4-(1-Azidoethyl)-4′-[3-(benzyloxy)phenoxy]-1,1′-biphenyl
(5e): Product was prepared according to the general procedure from
310 mg of 4e and obtained (266 mg, 80%).
Fig. 3. Effects of compound 1n on malonyl-CoA concentration in HCT-116
xenograft tumors. h, 40–49%.
1H NMR (300 MHz, DMSO‑d6) δ 0.99 (3H, t, J = 7.4 Hz), 1.60–1.82
(2H, m), 2.60 (3H, s), 3.86–3.99 (2H, m), 6.90–7.10 (6H, m), 7.69–7.85
(4H, m), 8.00 (2H, s). LCMS m/z calcd for C23H22O3: 346.16, found
347.1 [M + H] + .
1H NMR (300 MHz, DMSO‑d6) δ 1.49 (3H, d, J = 6.8 Hz), 4.89 (1H, q,
J = 6.8 Hz), 5.10 (2H, s), 6.62 (1H, ddd, J = 8.1, 2.3, 0.8 Hz), 6.72 (1H, t,
J = 2.3 Hz), 6.83 (1H, ddd, J = 8.3, 2.4, 0.8 Hz), 7.05–7.13 (2H, m),
7.26–7.52 (8H, m), 7.65–7.74 (4H, m).
1-{4′-[4-(2-Methoxyethyl)phenoxy][1,1′-biphenyl]-4-yl}
ethan-1-one (4c): Product was prepared according to the general pro-
cedure from 0.58 g of 4-(2-methoxyethyl)phenol (3c) and obtained
(0.29 g, 23%) as a white solid.
1H NMR (300 MHz, DMSO‑d6) δ 2.60 (3H, s), 2.73–2.86 (2H, m),
3.26 (3H, s), 3.46–3.57 (2H, m), 6.92–7.13 (4H, m), 7.28 (2H, d, J = 8.6
Hz), 7.78 (4H, dd, J = 10.7, 8.6 Hz), 8.03 (2H, d, J = 8.5 Hz). LCMS m/z
calcd for C23H22O3: 346.16, found 347.1 [M + H] + .
N-{1-[4′-(4-Propylphenoxy)[1,1′-biphenyl]-4-yl]ethyl}acet-
amide (1a): A mixture of 5a (190 mg, 0.53 mmol) and 10% Pd on
carbon (50% wet, 57 mg, 0.05 mmol) in THF (5 mL) was stirred at room
temperature for 2 h under H2 atmosphere (1 atm). The mixture was
filtered through a pad of Celite and concentrated under reduced pressure
to afford the amine. A mixture of amine and Ac2O (0.5 mL, 5.3 mmol) in
pyridine (2 mL) was stirred at room temperature for 30 min. The mixture
was concentrated under reduced pressure. The residue was purified by
column chromatography (NH silica gel, eluent: 10/90 to 10/0 AcOEt/
hexane) and crystallized from AcOEt and hexane to afford 1a (31 mg,
16%) as a white crystal.
1-{4′-[3-(2-Methoxyethyl)phenoxy][1,1′-biphenyl]-4-yl}
ethan-1-one (4d): Product was prepared according to the general pro-
cedure from 1 g of 3-(2-methoxyethyl)phenol (3d) and obtained (0.9 g,
40%) as a white solid.
1H NMR (300 MHz, DMSO‑d6) δ 2.61 (3H, s), 2.82 (2H, t, J = 6.8 Hz),
3.33 (3H, s), 3.54 (2H, t, J = 6.8 Hz), 6.91 (1H, dd, J = 7.7, 2.1 Hz), 6.99
(1H, s), 7.03–7.15 (3H, m), 7.27–7.39 (1H, m), 7.80 (4H, dd, J = 9.6, 8.8
Hz), 8.03 (2H, d, J = 8.5 Hz). LCMS m/z calcd for C23H22O3: 346.16,
found 347.1 [M + H] + .
1H NMR (300 MHz, DMSO‑d6) δ 0.90 (3H, t, J = 7.3 Hz), 1.36 (3H, d,
J = 7.0 Hz), 1.53–1.69 (2H, m), 1.85 (3H, s), 2.52–2.59 (2H, m),
4.80–5.02 (1H, m), 6.90–7.09 (4H, m), 7.22 (2H, d, J = 8.6 Hz), 7.36
(2H, d, J = 8.2 Hz), 7.50–7.69 (4H, m), 8.29 (1H, d, J = 8.1 Hz). 13C
1-{4′-[3-(Benzyloxy)phenoxy][1,1′-biphenyl]-4-yl}ethan-1-
7

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
NMR (75 MHz, DMSO‑d6) δ 14.08, 22.86, 23.14, 24.64, 36.97, 47.95,
118.91 (2C), 119.37 (2C), 126.77 (2C), 127.03 (2C), 128.57 (2C),
130.29 (2C), 135.37, 138.01, 138.38, 144.20, 154.74, 157.20, 168.68.
LCMS m/z calcd for C25H27NO2: 373.20, found 374.2 [M + H] + .
N-{1-[4′-(4-Propoxyphenoxy)[1,1′-biphenyl]-4-yl]ethyl}acet-
amide (1b): Product was prepared according to the general procedure
from 321 mg of 5b and obtained (115 mg, 34%) as a white crystal.
1H NMR (300 MHz, DMSO‑d6) δ 0.98 (3H, t, J = 7.4 Hz), 1.35 (3H, d,
J = 7.0 Hz), 1.62–1.78 (2H, m), 1.84 (3H, s), 3.92 (2H, t, J = 6.5 Hz),
4.83–5.01 (1H, m), 6.89–7.08 (6H, m), 7.35 (2H, d, J = 8.2 Hz), 7.58
(4H, dd, J = 15.7, 8.5 Hz), 8.21–8.37 (1H, m). 13C NMR (75 MHz,
DMSO‑d6) δ 10.89, 22.54, 22.86, 23.14, 47.95, 69.80, 116.14 (2C),
118.02 (2C), 121.32 (2C), 126.73 (2C), 127.01 (2C), 128.49 (2C),
134.88, 138.41, 144.13, 149.61, 155.67, 158.10, 168.68. LCMS m/z
calcd for C25H27NO3: 389.50, found 390.2 [M + H] + .
mL, 0.47 mmol) and K2CO3 (60 mg, 0.43 mmol) in DMF (2 mL) was
stirred at 80 ◦C for 2 h. After cooling to room temperature, the mixture
was extracted with AcOEt and water. The organic layer was washed with
sat. aq. NaCl, dried over MgSO4 and concentrated under reduced pres-
sure. The residue was purified by column chromatography (NH silica
gel, eluent: 10/90 to 100/0 AcOEt/hexane) and crystallized from AcOEt
and hexane to afford 1f (35 mg, 40%) as a white crystalline solid.
1H NMR (300 MHz, DMSO‑d6) δ 0.92 (3H, t, J = 7.4 Hz), 1.36 (3H, d,
J = 7.0 Hz), 1.39–1.47 (2H, m), 1.62–1.72 (2H, m), 1.85 (3H, s), 3.95
(2H, t, J = 6.4 Hz), 4.87–4.99 (1H, m), 6.55–6.63 (2H, m), 6.73 (1H, d, J
= 8.8 Hz), 7.09 (2H, d, J = 8.7 Hz), 7.28 (1H, t, J = 8.2 Hz), 7.37 (2H, d,
J = 8.3 Hz), 7.59 (2H, d, J = 8.2 Hz), 7.66 (2H, d, J = 8.8 Hz), 8.31 (1H,
d, J = 7.6 Hz). 13C NMR (75 MHz, DMSO‑d6) δ 14.14, 19.18, 22.86,
23.15, 31.14, 47.95, 67.79, 105.47, 110.01, 110.99, 119.57 (2C),
126.76 (2C), 127.03 (2C), 128.60 (2C), 131.04, 135.69, 138.24, 144.28,
156.49, 158.10, 160.56, 168.69. LCMS m/z calcd for C26H29NO3:
403.21, found 404.1 [M + H] + . Anal. Calcd for C26H29NO3: C, 77.39;
H, 7.24; N, 3.47. Found: C, 77.09; H, 7.05; N, 3.24. Mp 108–110 ◦C.
N-(1-{4′-[3-(Cyclopropylmethoxy)phenoxy][1,1′-biphenyl]-4-
yl}ethyl)acetamide (1 g): Product was prepared according to the
general procedure of 1f from 75 mg of 1e and obtained (42 mg, 49%) as
a white crystalline solid.
N-(1-{4′-[4-(2-Methoxyethyl)phenoxy][1,1′-biphenyl]-4-yl}
ethyl)acetamide (1c): Product was prepared according to the general
procedure from 200 mg of 5c and obtained (11 mg, 5%) as a white
crystalline solid.
1H NMR (300 MHz, DMSO‑d6) δ 1.36 (3H, d, J = 7.0 Hz), 1.84 (3H,
s), 2.74–2.85 (2H, m), 3.25 (3H, s), 3.54 (2H, t, J = 6.8 Hz), 4.84–5.02
(1H, m), 6.91–7.10 (4H, m), 7.23–7.31 (2H, m), 7.32–7.39 (2H, m),
7.52–7.71 (4H, m), 8.19–8.39 (1H, m). LCMS m/z calcd for C25H27NO3:
389.50, found 390.2 [M + H] + .
1H NMR (300 MHz, DMSO‑d6) δ 0.26–0.35 (2H, m), 0.50–0.59 (2H,
m), 1.12–1.27 (1H, m), 1.36 (3H, d, J = 7.0 Hz), 1.85 (3H, s), 3.80 (2H,
d, J = 7.0 Hz), 4.93 (1H, t, J = 7.5 Hz), 6.55–6.63 (2H, m), 6.68–6.76
(1H, m), 7.08 (2H, d, J = 8.7 Hz), 7.28 (1H, t, J = 8.3 Hz), 7.37 (2H, d, J
= 8.2 Hz), 7.59 (2H, d, J = 8.2 Hz), 7.66 (2H, d, J = 8.8 Hz), 8.31 (1H, d,
J = 8.2 Hz). 13C NMR (75 MHz, DMSO‑d6) δ 3.54 (2C), 10.53, 22.85,
23.15, 47.96, 72.67, 105.78, 110.23, 111.07, 119.46 (2C), 126.82 (2C),
127.04 (2C), 128.60 (2C), 130.97, 135.78, 138.33, 144.27, 156.54,
158.14, 160.57, 168.69. LCMS m/z calcd for C26H27NO3: 401.20, found
402.2 [M + H] + . Anal. Calcd for C26H27NO3: C, 77.78; H, 6.78; N, 3.49.
Found: C, 77.90; H, 6.53; N, 3.19. Mp 122–124 ◦C
N-{1-[4′-(3-Hydroxyphenoxy)[1,1′-biphenyl]-4-yl]ethyl}acet-
amide (1e): Product was prepared according to the general procedure
from 266 mg of 5e and obtained (150 mg, 68%) as a white crystal.
1H NMR (300 MHz, DMSO‑d6) δ 1.36 (3H, d, J = 7.0 Hz), 1.85 (3H,
s), 4.93 (1H, quin, J = 7.2 Hz), 6.38–6.43 (1H, m), 6.47 (1H, dd, J = 8.1,
1.6 Hz), 6.55 (1H, dd, J = 8.1, 1.5 Hz), 7.08 (2H, d, J = 8.7 Hz), 7.17
(1H, t, J = 8.1 Hz), 7.37 (2H, d, J = 8.3 Hz), 7.58 (2H, d, J = 8.2 Hz),
7.65 (2H, d, J = 8.7 Hz), 8.32 (1H, d, J = 8.0 Hz), 9.63 (1H, s). LCMS m/z
calcd for C22H21NO3: 347.15, found 348.1 [M + H] + .
5.1.2. N-(1-{4′-[3-(2-Methoxyethyl)phenoxy][1,1′-biphenyl]-4-yl}ethyl)
acetamide (1d)
5.1.3. N-[1-(4-{4-[3-(Cyclopropylmethoxy)phenoxy]phenoxy}phenyl)
ethyl]acetamide (1 h)
1-{4′-[3-(2-Methoxyethyl)phenoxy][1,1′-biphenyl]-4-yl}
ethan-1-amine (6d): A mixture of 5d (970 mg, 2.6 mmol) and Ph3P
(1.4 g, 5.3 mmol) in THF (6 mL) and water (3 mL) was stirred at 60 ◦C for
1 h. After cooling to room temperature, the mixture was extracted with
AcOEt and water. The organic layer was washed with sat. aq. NaCl, dried
over MgSO4 and concentrated under reduced pressure. The residue was
purified by column chromatography (silica gel, eluent: 0/100 to 75/25
MeOH/AcOEt) to afford 6d (561 mg, 62%).
1-[4-(4-Iodophenoxy)phenyl]ethan-1-one (9): A mixture of 4-
iodophenol (7) (1.7 g, 7.6 mmol), 1-(4-fluorophenyl)ethan-1-one (8)
(1 g, 7.2 mmol) and K2CO3 (1.2 g, 8.7 mmol) in DMF (6 mL) was stirred
◦
at 50 C overnight. The mixture was diluted with AcOEt and toluene,
washed with 0.1 N HCl aq., sat. aq. NaCl and concentrated under
reduced pressure. The residue was purified by column chromatography
(silica gel, eluent: 5/95 to 50/50 AcOEt/Hexane) to give 9 (1.7 g, 69%)
as a yellow amorphous powder.
1H NMR (300 MHz, DMSO‑d6) δ 1.27 (3H, d, J = 6.6 Hz), 1.90–2.00
(2H, m), 2.81 (2H, t, J = 6.8 Hz), 3.23 (3H, s), 3.53 (2H, t, J = 6.8 Hz),
3.97–4.07 (1H, m), 6.87 (1H, ddd, J = 8.1, 2.5, 0.8 Hz), 6.96 (1H, t, J =
1.9 Hz), 7.01–7.09 (3H, m), 7.27–7.35 (1H, m), 7.40–7.47 (2H, m),
7.53–7.60 (2H, m), 7.62–7.69 (2H, m). LCMS m/z calcd for C23H25NO2:
347.45, found 331.2 [M + H–NH3] + .
1H NMR (300 MHz, CDCl3) δ 2.58 (3H, s), 6.79–6.87 (2H, m),
6.97–7.03 (2H, m), 7.66–7.71 (2H, m), 7.93–7.98 (2H, m). LCMS m/z
calcd for C14H11IO2: 337.98, found 338.8 [M + H] + .
1-(4-{4-[3-(Cyclopropylmethoxy)phenoxy]phenoxy}phenyl)
ethan-1-one (11): A mixture of 3-(cyclopropylmethoxy)phenol (10)
(80 mg, 0.49 mmol), CuI (25.3 mg, 0.13 mmol), dimethylaminoacetic
acid hydrochloride (56 mg, 0.4 mmol), Cs2CO3 (217 mg, 0.67 mmol)
and DME (1 mL) was stirred at 90 ◦C overnight. The reaction mixture
was diluted with AcOEt, filtered through a pad of Celite and washed
with AcOEt. The filtrate was concentrated under reduced pressure. The
residue was diluted with toluene and AcOEt, filtered through a pad of
silica gel and washed with 50% AcOEt in hexane. The filtrate was
concentrated under reduced pressure. The residue was purified by col-
umn chromatography (silica gel, eluent: 0/100 to 20/80 AcOEt/hexane)
to give 11 (93 mg, 56%) as a colorless oil.
N-(1-{4′-[3-(2-Methoxyethyl)phenoxy][1,1′-biphenyl]-4-yl}
ethyl)acetamide (1d): A mixture of 6d (561 mg, 1.6 mmol) and Ac2O
(0.75 mL, 8.0 mmol) in pyridine (4 mL) was stirred at room temperature
for 30 min. The mixture was concentrated under reduced pressure. The
residue was purified by column chromatography (NH silica gel, eluent:
10/90 to 100/0 AcOEt/hexane) to afford 1d (616 mg, 98%) as a white
amorphous.
1H NMR (300 MHz, DMSO‑d6) δ 1.36 (3H, d, J = 7.0 Hz), 1.85 (3H,
s), 2.81 (2H, t, J = 6.7 Hz), 3.23 (3H, s), 3.53 (2H, t, J = 6.8 Hz), 4.93
(1H, quin, J = 7.1 Hz), 6.88 (1H, dd, J = 8.1, 1.7 Hz), 6.97 (1H, s),
7.01–7.10 (3H, m), 7.27–7.41 (3H, m), 7.58 (2H, d, J = 8.3 Hz), 7.65
(2H, d, J = 8.7 Hz), 8.31 (1H, d, J = 8.0 Hz). LCMS m/z calcd for
1H NMR (300 MHz, CDCl3) δ 0.22–0.42 (2H, m), 0.48–0.72 (2H, m),
1.22–1.30 (1H, m), 2.58 (3H, s), 3.78 (2H, d, J = 7.0 Hz), 6.42–6.72 (3H,
m), 6.90–7.10 (6H, m), 7.18–7.25 (1H, m), 7.90–7.99 (2H, m).
N-[1-(4-{4-[3-(Cyclopropylmethoxy)phenoxy]phenoxy}
C
25H27NO3: 389.50, found 390.1 [M + H] + . Anal Calcd for C25H27NO3:
C, 77.09; H, 6.99; N, 3.60. Found: C, 76.85; H, 6.82; N, 3.55.
N-{1-[4′-(3-Butoxyphenoxy)[1,1′-biphenyl]-4-yl]ethyl}acet-
amide (1f): A mixture of 1e (75 mg, 0.22 mmol), 1-bromobutane (0.05
phenyl)ethyl]acetamide (1 h): NaBH3CN (39 mg, 0.62 mmol) was
added to a solution of ammonium acetate (329 mg, 4.3 mmol) and 11
(80 mg, 0.21 mmol) in MeOH (5 mL) at room temperature. The mixture
8

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
was stirred at 60 ◦C overnight. After cooling, the mixture was concen-
trated under reduced pressure and diluted with sat. aq. NaHCO3 and
AcOEt. The mixture was extracted with AcOEt. The organic layer was
separated, washed with sat. aq. NaCl, dried over MgSO4 and concen-
trated under reduced pressure to give the amine. A mixture of the amine,
Et3N (0.15 mL, 1.1 mmol) and Ac2O (0.059 mL, 0.6 mmol) in THF (1 mL)
was stirred at room temperature for 3 h. The mixture was quenched with
concentrated under reduced pressure. The residue was purified by col-
umn chromatography (silica gel, eluent: 10/90 to 100/0 AcOEt/Hex-
ane) and crystallized from IPE and hexane to give 1i (125 mg, 40%) as a
white solid.
1H NMR (300 MHz, CDCl3) δ 0.27–0.40 (2H, m), 0.62–0.70 (2H, m),
1.22–1.32 (1H, m), 1.50 (3H, d, J = 6.9 Hz), 1.99 (3H, s), 3.78 (2H, d, J
= 6.9 Hz), 5.03 (2H, s), 5.06–5.18 (1H, m), 5.56–5.70 (1H, m),
6.54–6.71 (3H, m), 6.93–7.00 (2H, m), 7.02–7.07 (2H, m), 7.19–7.31
(3H, m), 7.40 (2H, d, J = 8.6 Hz). 13C NMR (75 MHz, DMSO‑d6) δ 3.53
(2C), 10.52, 22.92, 23.15, 47.54, 69.21, 72.64, 105.66, 110.17, 110.94,
114.93 (2C), 119.09 (2C), 127.61 (2C), 130.02 (2C), 130.94, 132.74,
137.49, 156.60, 157.53, 158.20, 160.55, 168.51. LCMS m/z calcd for
◦
water at 0 C and extracted with AcOEt. The organic layer was sepa-
rated, washed with sat. aq. NaCl, dried over MgSO4 and concentrated
under reduced pressure. The residue was purified by column chroma-
tography (silica gel, eluent: 0/100 to 100/0 AcOEt/hexane) to give 1 h
(60 mg, 68%) as a white solid.
1H NMR (300 MHz, CDCl3) δ 0.27–0.38 (2H, m), 0.60–0.70 (2H, m),
1.21–1.31 (1H, m), 1.49 (3H, d, J = 7.0 Hz), 1.99 (3H, s), 3.77 (2H, d, J
= 6.8 Hz), 5.02–5.19 (1H, m), 5.55–5.67 (1H, m), 6.51–6.66 (3H, m),
6.94–7.05 (6H, m), 7.21 (1H, t, J = 8.1 Hz), 7.27–7.31 (2H, m). LCMS m/
z calcd for C26H27NO4: 179.09, found 201.9 [M + H + Na]+.
C27H29NO4: 431.21, found 432.1 [M + H] + .
5.1.5. N-{1-[4-({6-[3-(Cyclopropylmethoxy)phenoxy]pyridin-3-yl}
methoxy)phenyl]ethyl}acetamide (1j)
6-[3-(Cyclopropylmethoxy)phenoxy]pyridine-3-carbaldehyde
(15b): A mixture of 10 (1 g, 6.1 mmol), 6-chloropyridine-3-carbalde-
hyde (14b) (0.86 g, 6.1 mmol) and K2CO3 (1.7 g, 12.2 mmol) in DMF
(20 mL) was stirred at 60 ◦C for 2 h. After cooling to room temperature,
the mixture was extracted with AcOEt and water. The organic layer was
washed with sat. aq. NaCl, dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by column chromatography
(silica gel, eluent: 5/95 to 25/75 AcOEt/hexane) to afford 15b (875 mg,
53%).
5.1.4. N-{1-[4-({4-[3-(Cyclopropylmethoxy)phenoxy]phenyl}methoxy)
phenyl]ethyl}acetamide (1i)
N-[1-(4-Hydroxyphenyl)ethyl]acetamide (13): A mixture of 4-(1-
aminoethyl)phenol (12) (3 g, 22 mmol) and Ac2O (2.1 mL, 22 mmol) in
THF (20 mL) was stirred at room temperature for 2 h. The mixture was
quenched with sat. aq. NH4Cl at room temperature and extracted with
AcOEt. The organic layer was separated, washed with sat. aq. NaCl,
dried over MgSO4 and concentrated under reduced pressure. The residue
was purified by column chromatography (silica gel, eluent: 0/100 to
100/0 AcOEt/hexane and 0/100 to 30/70 MeOH/AcOEt) to give 13
(2.4 g, 61%) as a white solid.
LCMS m/z calcd for C16H15NO3: 269.11, found 269.9 [M + H] + .
{6-[3-(Cyclopropylmethoxy)phenoxy]pyridin-3-yl}methanol
(16b): To a solution of 15b (875 mg, 3.3 mmol) in MeOH (10 mL) was
added NaBH4 (123 mg, 3.3 mmol) at 0 ◦C. The mixture was stirred at 0
◦C for 2 h. The mixture was quenched with water at 0 ◦C and extracted
with AcOEt/THF. The organic layer was separated, washed with sat. aq.
NaCl, dried over MgSO4 and concentrated under reduced pressure. The
residue was purified by column chromatography (silica gel, eluent: 10/
90 to 100/0 AcOEt/hexane) to give 16b (264 mg, 30%).
1H NMR (300 MHz, CD3OD) δ 1.39 (3H, d, J = 7.0 Hz), 1.93 (3H, s),
4.89–4.97 (1H, m), 6.73 (2H, d, J = 8.5 Hz), 7.13 (2H, d, J = 8.7 Hz).
LCMS m/z calcd for C10H13NO2: 179.22, found 440.0 [M + H + Na]+.
Ethyl 4-[3-(cyclopropylmethoxy)phenoxy]benzoate (15a): A
mixture of 10 (5 g, 31 mmol), ethyl 4-fluorobenzoate (14a) (5 g, 30
mmol) and Cs2CO3 (15 g, 46 mmol) in DMF (50 mL) was stirred at 100
◦C overnight. After cooling to room temperature, the mixture was
extracted with AcOEt and water. The organic layer was washed with sat.
aq. NaCl, dried over MgSO4 and concentrated under reduced pressure.
The residue was purified by column chromatography (silica gel, eluent:
0/100 to 10/90 AcOEt/hexane) to afford 15a (7.1 g, 75%).
LCMS m/z calcd for C16H17NO3: 271.12, found 271.9 [M + H] + .
N-{1-[4-({6-[3-(Cyclopropylmethoxy)phenoxy]pyridin-3-yl}
methoxy)phenyl]ethyl}acetamide (1j): To a solution of 16b (130 mg,
0.48 mmol) and Et3N (0.16 mL, 1.2 mmol) in THF (4 mL) was added
MsCl (0.045 mL, 0.57 mmol) at 0 ◦C. The mixture was stirred at 0 ◦C for
4 h. The mixture was quenched with water at 0 ◦C and extracted with
AcOEt. The organic layer was separated, washed with sat. aq. NaCl,
dried over MgSO4 and concentrated under reduced pressure to afford the
metanesulfonate. A mixture of the methanesulfonate, 13 (103 mg, 0.58
mmol) and K2CO3 (100 mg, 0.72 mmol) in DMF (7 mL) was stirred at 50
◦C overnight. The mixture was quenched with water at room tempera-
ture and extracted with AcOEt. The organic layer was separated, washed
with sat. aq. NaCl, dried over MgSO4 and concentrated under reduced
pressure. The residue was purified by column chromatography (silica
gel, eluent: 10/90 to 100/0 AcOEt/hexane) to give 1j (32 mg, 15%) as a
light brown oil.
1H NMR (300 MHz, DMSO‑d6) δ 0.26–0.34 (2H, m), 0.50–0.60 (2H,
m), 1.19–1.25 (1H, m), 1.31 (3H, t, J = 7.1 Hz), 3.80 (2H, d, J = 7.0 Hz),
4.29 (2H, q, J = 7.1 Hz), 6.60–6.70 (2H, m), 6.80 (1H, ddd, J = 8.3, 2.3,
0.9 Hz), 7.01–7.13 (2H, m), 7.33 (1H, t, J = 8.3 Hz), 7.91–8.01 (2H, m).
LCMS m/z calcd for C19H20O4: 312.14, found 313.1 [M + H] + .
{4-[3-(Cyclopropylmethoxy)phenoxy]phenyl}methanol (16a):
To an ice cold mixture of LiAlH4 (0.13 g, 3.4 mmol) in THF (10 mL) was
added 15a (1 g, 3.2 mmol) in THF (5 mL) dropwise. After stirring at 0 ◦C
for 30 min, water (0.13 mL) was added slowly followed by 1 N NaOH
(0.13 mL). Water (0.39 mL) was added and the mixture was stirred at
room temperature for 30 min. The mixture was filtered through a pad of
Celite and concentrated under reduced pressure to afford 16a (0.85 g,
98%).
1H NMR (300 MHz, CDCl3) δ 0.22–0.32 (2H, m), 0.50–0.61 (2H, m),
1.13–1.24 (1H, m), 1.39 (3H, d, J = 7.0 Hz), 1.90 (3H, s), 3.71 (2H, d, J
= 7.0 Hz), 4.92 (2H, s), 4.96–5.08 (1H, m), 5.64 (1H, d, J = 7.7 Hz),
6.58–6.72 (3H, m), 6.82–6.90 (3H, m), 7.16–7.23 (3H, m), 7.69 (1H, dd,
J = 8.4, 2.5 Hz), 8.16 (1H, d, J = 2.1 Hz). LCMS m/z calcd for
C26H28N2O4: 432.20, found 433.1 [M + H] + .
1H NMR (300 MHz, DMSO‑d6) δ 0.21–0.38 (2H, m), 0.47–0.65 (2H,
m), 1.17–1.24 (1H, m), 3.77 (2H, d, J = 7.0 Hz), 4.47 (2H, d, J = 5.7 Hz),
5.16 (1H, t, J = 5.7 Hz), 6.45–6.54 (2H, m), 6.61–6.75 (1H, m),
6.89–7.04 (2H, m), 7.17–7.28 (1H, m), 7.33 (2H, d, J = 8.7 Hz). LCMS
m/z calcd for C17H18O3: 270.13, found 253.1 [M + H–H2O]+.
N-{1-[4-({4-[3-(Cyclopropylmethoxy)phenoxy]phenyl}
5.1.6. N-{1-[4-({4-[3-(Cyclopropylmethoxy)phenoxy]-2-fluorophenyl}
methoxy)phenyl]ethyl}acetamide (1 l)
methoxy)phenyl]ethyl}acetamide (1i): 16a (200 mg, 0.74 mmol)
and 13 (146 mg, 0.81 mmol) were dissolved in toluene (4 mL). To the
mixture were added tributylphosphine (0.3 mL, 1.2 mmol) followed by
1,1′-(azodicarbonyl)-dipiperidine (303 mg, 1.2 mmol) at room temper-
ature. The mixture was stirred at room temperature overnight. To the
solution was added AcOEt/hexane (1/2) (20 mL) and white precipitate
was filtered off and washed with 33% AcOEt/hexane. The filtrate was
4-[3-(Cyclopropylmethoxy)phenoxy]-2-fluorobenzaldehyde
(15c): A mixture of 10 (849 mg, 5.2 mmol), 2,4-difluorobenzaldehyde
(14c) (700 mg, 4.9 mmol) and Cs2CO3 (1.6 g, 4.9 mmol) in DMF (10
mL) was stirred at 60 ◦C for 3 h. The mixture was quenched with water at
0 ◦C and extracted with AcOEt. The organic layer was separated, washed
with sat. aq. NaCl, dried over MgSO4 and concentrated under reduced
pressure. The residue was purified by column chromatography (silica
9

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
gel, eluent: 0/100 to 50/50 AcOEt/hexane) to give 15c (852 mg, 60%).
LCMS m/z calcd for C17H15FO3: 286.10, found 286.9 [M + H] + .
N-{1-[4-({4-[3-(Cyclopropylmethoxy)phenoxy]-2-fluo-
washed with sat. aq. NaCl, dried over MgSO4 and concentrated under
reduced pressure. To the residue was added toluene (10 mL). To the
mixture were added DPPA (1.5 g, 5.5 mmol) and DBU (0.8 mL, 5.3
mmol). After stirring at room temperature for 2 h, the mixture was
extracted with toluene and water. The organic layer was washed with
sat. aq. NaCl, dried over MgSO4 and concentrated under reduced pres-
sure. The residue was purified by column chromatography (silica gel,
eluent: 5/95 to 25/75 AcOEt/hexane) to afford 21 (0.94 g, 88%).
1H NMR (300 MHz, DMSO‑d6) δ 0.22–0.39 (2H, m), 0.49–0.62 (2H,
m), 1.20–1.27 (1H, m), 1.43 (3H, d, J = 6.8 Hz), 3.80 (2H, d, J = 7.0 Hz),
4.77 (1H, q, J = 6.8 Hz), 5.17 (2H, s), 6.55–6.67 (2H, m), 6.72–6.80 (1H,
m), 7.01–7.11 (2H, m), 7.29 (2H, s), 7.48 (2H, dd, J = 8.0, 2.0 Hz),
7.52–7.59 (1H, m), 8.37 (1H, d, J = 2.4 Hz). LCMS m/z calcd for
rophenyl}methoxy)phenyl]ethyl}acetamide (1 l): 15c (300 mg, 1.1
mmol) were dissolved in MeOH (5 mL). NaBH4 (119 mg, 3.1 mmol) was
added to the mixture at 0 ◦C. The mixture was stirred at 0 ◦C for 3 h. The
mixture was quenched with water at 0 ◦C and extracted with AcOEt. The
organic layer was separated, washed with sat. aq. NaCl, dried over
MgSO4 and concentrated under reduced pressure to afford the alcohol.
The alcohol and 13 (282 mg, 1.6 mmol) were dissolved in toluene (8
mL). To the mixture was added tributylphosphine (0.393 mL, 1.6 mmol)
followed by 1,1′-(azodicarbonyl)-dipiperidine (397 mg, 1.6 mmol) at
room temperature. The mixture was stirred at room temperature over-
night. To the solution was added 33% AcOEt/hexane and white pre-
cipitate was filtered off and washed with 33% AcOEt/hexane. The
filtrate was concentrated under reduced pressure. The residue was pu-
rified by column chromatography (silica gel, eluent: 10/90 to 100/
0 AcOEt/hexane) and by preparative HPLC. The residue was crystallized
from IPE and hexane to give 1 l (142 mg, 30%) as a white crystalline
solid.
C24H24N4O3: 416.18, found 417.2 [M + H] + .
1-[4-({5-[3-(Cyclopropylmethoxy)phenoxy]pyridin-2-yl}
methoxy)phenyl]ethan-1-amine (22): A mixture of 21 (937 mg, 2.3
mmol) and Ph3P (1.2 g, 4.6 mmol) in THF (6 mL) and water (3 mL) was
stirred at 60 ◦C for 1 h. After cooling to room temperature, the mixture
was extracted with AcOEt and water. The organic layer was washed with
sat. aq. NaCl, dried over MgSO4 and concentrated under reduced pres-
sure. The residue was purified by column chromatography (silica gel,
eluent: 0/100 to 100/0 MeOH/AcOEt) to afford 22 (693 mg, 79%).
1H NMR (300 MHz, DMSO‑d6) δ 0.26–0.34 (2H, m), 0.52–0.60 (2H,
m), 1.13–1.15 (1H, m), 1.21 (3H, d, J = 4.8 Hz), 1.82 (2H, brs), 3.80
(2H, d, J = 7.0 Hz), 3.93 (1H, q, J = 6.5 Hz), 5.13 (2H, s), 6.55–6.66 (2H,
m), 6.72–6.79 (1H, m), 6.90–6.98 (2H, m), 7.24–7.33 (3H, m),
7.43–7.49 (1H, m), 7.50–7.56 (1H, m), 8.37 (1H, d, J = 2.2 Hz). LCMS
m/z calcd for C24H26N2O3: 390.19, found 391.1 [M + H] + .
1H NMR (300 MHz, CD3OD) δ 0.28–0.37 (2H, m), 0.54–0.64 (2H, m),
1.14–1.28 (1H, m), 1.41 (3H, d, J = 7.0 Hz), 1.94 (3H, s), 3.79 (2H, d, J
= 6.8 Hz), 4.91–5.01 (1H, m), 5.06 (2H, s), 6.55–6.62 (2H, m),
6.68–6.83 (3H, m), 6.91–6.99 (2H, m), 7.19–7.32 (3H, m), 7.41–7.50
(1H, m), 8.36 (1H, d, J = 7.9 Hz). 13C NMR (75 MHz, DMSO‑d6) δ 3.54
(2C), 10.51, 22.94, 23.15, 47.54, 63.70 (d, J = 2.25 Hz), 72.72, 106.26
(d, J = 24 Hz), 106.30, 111.10, 111.58, 114.40, 114.86 (2C), 119.07 (d,
J = 15 Hz), 127.63 (2C), 131.12, 132.38, 137.72, 157.17, 157.39,
158.65 (d, J = 11.25 Hz), 160.62, 161.44 (d, J = 246 Hz), 168.51. LCMS
m/z calcd for C27H28FNO4: 449.20, found 450.1 [M + H] + . Anal. Calcd
for C27H28FNO4: C,72.14; H,6.28; N,3.12. Found: C,71.90; H,6.30;
N,3.10.
N-{1-[4-({5-[3-(Cyclopropylmethoxy)phenoxy]pyridin-2-yl}
methoxy)phenyl]ethyl}acetamide (1 k): A mixture of 22 (693 mg,
1.8 mmol) and Ac2O (0.75 mL, 8.0 mmol) in pyridine (4 mL) was stirred
at room temperature for 30 min. The mixture was concentrated under
reduced pressure. The residue was purified by column chromatography
(NH silica gel, eluent: 10/90 to 100/0 AcOEt/hexane) to afford 1 k (585
mg, 76%).
5.1.7. N-{1-[4-({5-[3-(Cyclopropylmethoxy)phenoxy]pyridin-2-yl}
methoxy)phenyl]ethyl}acetamide (1 k)
1-{4-[(5-Bromopyridin-2-yl)methoxy]phenyl}ethan-1-one (19):
A mixture of (5-bromopyridin-2-yl)methanol (17) (1 g, 5.3 mmol), 1-(4-
hydroxyphenyl)ethan-1-one (18) (0.7 g, 5.1 mmol), DIAD (40% in
toluene, 3 mL, 5.7 mmol) and Ph3P (1.5 g, 5.7 mmol) in THF (15 mL)
was stirred at room temperature overnight. The mixture was concen-
trated under reduced pressure and the residue was triturated with EtOH
to afford 19 (1.4 g, 88%) as a white solid.
1H NMR (300 MHz, DMSO‑d6) δ 0.26–0.34 (2H, m), 0.50–0.60 (2H,
m), 1.17–1.24 (1H, m), 1.30 (3H, d, J = 7.0 Hz), 1.81 (3H, s), 3.80 (2H,
d, J = 7.0 Hz), 4.85 (1H, quin, J = 7.1 Hz), 5.13 (2H, s), 6.56–6.66 (2H,
m), 6.75 (1H, dd, J = 8.3, 1.7 Hz), 6.96 (2H, d, J = 8.7 Hz), 7.22 (2H, d,
J = 8.6 Hz), 7.29 (1H, t, J = 8.2 Hz), 7.43–7.49 (1H, m), 7.50–7.55 (1H,
m), 8.20 (1H, d, J = 8.1 Hz), 8.36 (1H, d, J = 2.2 Hz). 13C NMR (75 MHz,
DMSO‑d6) δ 3.55 (2C), 10.54, 22.94, 23.15, 47.54, 66.86, 72.69, 108.03,
111.29, 111.73, 113.48, 114.98 (2C), 127.62 (2C), 128.33, 130.57,
137.70, 140.66, 147.56, 155.42, 157.31, 160.29, 163.25, 168.53. LCMS
m/z calcd for C26H28N2O4: 432.20, found 433.4 [M + H] + .
1H NMR (300 MHz, DMSO‑d6) δ 2.51 (3H, s), 5.27 (2H, s), 7.08–7.17
(2H, m), 7.51 (1H, d, J = 8.2 Hz), 7.94 (2H, d, J = 9.0 Hz), 8.11 (1H, dd,
J = 8.4, 2.4 Hz), 8.73 (1H, d, J = 2.0 Hz). LCMS m/z calcd for
C
14H11BrNO2: 307.00, found 308.0 [M + H] + .
1-[4-({5-[3-(Cyclopropylmethoxy)phenoxy]pyridin-2-yl}
5.1.8. N-{1-[4-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-
methoxy)phenyl]ethan-1-one (20): A mixture of 19 (1.4 g, 4.7 mmol),
10 (0.8 g, 4.9 mmol), picolinic acid (0.12 g, 0.97 mmol), CuI (0.09 g,
0.47 mmol) and K3PO4 (1.5 g, 7.1 mmol) in DMSO (15 mL) was stirred at
90 ◦C for 4 h. After cooling to room temperature, the mixture was
extracted with AcOEt and water. The organic layer was washed with sat.
aq. NaCl, dried over MgSO4 and concentrated under reduced pressure.
The residue was purified by column chromatography (NH silica gel,
eluent: 5/95 to 25/75 AcOEt/hexane) to afford 20 (1 g, 55%).
difluorophenyl}methoxy)phenyl]ethyl}acetamide (1 m)
(2,6-Difluoro-4-iodophenyl)methanol (24): To an ice cold stirred
mixture of 2,6-difluoro-4-iodobenzaldehyde (23) (6.6 g, 25 mmol) in
EtOH (60 mL) was added NaBH4 (1 g, 26 mmol). After stirring at 0 ◦C for
1 h, the mixture was acidified with 1 N HCl and concentrated under
reduced pressure. The residue was extracted with AcOEt and water. The
organic layer was washed with sat. aq. NaCl, dried over MgSO4 and
concentrated under reduced pressure to afford 24 (6.5 g, 98%) as a
white solid.
1H NMR (300 MHz, DMSO‑d6) δ 0.26–0.34 (2H, m), 0.50–0.60 (2H,
m), 1.19–1.25 (1H, m), 2.52 (3H, s), 3.80 (2H, d, J = 7.0 Hz), 5.26 (2H,
s), 6.60 (1H, dt, J = 8.1, 1.2 Hz), 6.64 (1H, t, J = 2.3 Hz), 6.73–6.79 (1H,
m), 7.11–7.17 (2H, m), 7.30 (1H, t, J = 8.2 Hz), 7.44–7.52 (1H, m),
7.53–7.59 (1H, m), 7.89–7.99 (2H, m), 8.38 (1H, d, J = 2.2 Hz). LCMS
m/z calcd for C24H23NO4: 389.16, found 390.3 [M + H] + .
1H NMR (300 MHz, DMSO‑d6) δ 4.44 (2H, d, J = 5.7 Hz), 5.28 (1H, t,
J = 5.7 Hz), 7.50–7.58 (2H, m). LCMS m/z calcd for C7H5F2IO: 269.94,
found 253.0 [M + H–H2O]+.
N-(1-{4-[(2,6-Difluoro-4-iodophenyl)methoxy]phenyl}ethyl)
acetamide (25): To a mixture of 24 (4.7 g, 17 mmol), 13 (3.1 g, 17
mmol) and Ph3P (5 g, 19 mmol) in THF (80 mL) was added DIAD (3.84 g,
19.0 mmol) dropwise, and the mixture was stirred at 12 ◦C for 4 h under
N2 atmosphere. The reaction mixture was poured into water and
extracted with AcOEt. The organic layer was washed with sat. aq. NaCl,
dried over Na2SO4 and concentrated under reduced pressure. The
2-{[4-(1-Azidoethyl)phenoxy]methyl}-5-[3-(cyclo-
propylmethoxy)phenoxy]pyridine (21): To an ice cold mixture of 20
(1 g, 2.6 mmol) in EtOH (5 mL) and THF (5 mL) was added NaBH4 (0.1 g,
2.6 mmol). After stirring at room temperature for 4 h, the mixture was
acidified with 1 N HCl and extracted with AcOEt. The organic layer was
10

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
residue was purified by column chromatography (silica gel, eluent:
AcOEt) and by prep-HPLC (0.1% NH4HCO3 as additive) to afford 25 (6 g,
76%) as a white solid.
were added LiOH⋅H2O (100 mg, 4.2 mmol) and water (6 mL) at 30 ◦C.
The mixture was stirred at 26–32 ◦C for 12 h. After removal of MeOH
under reduced pressure, the mixture was diluted with water (30 mL).
The mixture was acidified with 1 N HCl, and was then extracted with
AcOEt. The organic layer was washed with sat. aq. NaCl, dried over
Na2SO4, and concentrated under reduced pressure to afford the car-
boxylic acid. To a solution of the carboxylic acid in DMF (10 mL) were
added N,O-dimethylhydroxylamine hydrochloride (191 mg, 2.6 mmol),
HOBt (437 mg, 3.2 mmol), EDCI (621 mg, 3.2 mmol) and Et3N (663 mg,
6.6 mmol) at 30 ◦C, and the mixture was stirred at 30 ◦C for 3 h. The
mixture was diluted with water and extracted with AcOEt. The com-
bined organic layers were washed with sat. aq. NaCl, dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified by
column chromatography (silica gel, eluent: 33/67 AcOEt/Petroleum
ether) to afford 29a (530 mg, 83%) as a colorless oil.
1H NMR (400 MHz, DMSO‑d6) δ 1.29 (3H, d, J = 6.8 Hz), 1.81 (3H,
s), 4.80–4.90 (1H, m), 5.03 (2H, s), 6.95 (2H, d, J = 8.8 Hz), 7.22 (2H, d,
J = 8.4 Hz), 7.65 (2H, d, J = 6.8 Hz), 8.21 (1H, d, J = 8.4 Hz). LCMS m/z
calcd for C17H16F2INO2: 431.02, found 431.8 [M + H] + .
N-{1-[4-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-difluor-
ophenyl}methoxy)phenyl]ethyl}acetamide (1 m): A mixture of 10
(318 mg, 1.9 mmol), 25 (700 mg, 1.6 mmol), CuI (75 mg, 0.39 mmol),
picolinic acid (160 mg, 1.3 mmol) and K3PO4 (690 mg, 3.3 mmol) in
DMSO (5 mL) was stirred at 90 ◦C overnight. After cooling to room
temperature, the mixture was extracted with AcOEt and water. The
organic layer was washed with sat. aq. NaCl, dried over MgSO4 and
concentrated in vacuo. The residue was purified by column chroma-
tography (NH silica gel, eluent: 10/90 to 100/0 AcOEt/hexane) to afford
1 m (292 mg, 38%).
1H NMR (400 MHz, CDCl3) δ 0.25–0.40 (2H, m), 0.55–0.73 (2H, m),
1.15–1.35 (1H, m), 3.38 (3H, s), 3.59 (3H, s), 3.78 (2H, d, J = 6.8 Hz),
5.42 (2H, s), 6.55 (2H, d, J = 8.8 Hz), 6.57–6.70 (2H, m), 6.71–6.80 (2H,
m), 7.20–7.35 (1H, m), 8.00 (1H, dd, J = 8.8, 2.4 Hz), 8.66 (1H, d, J =
2.0 Hz). LCMS m/z calcd for C25H24F2N2O5: 470.47, found 471.0 [M +
H] + .
1H NMR (300 MHz, DMSO‑d6) δ 0.27–0.35 (2H, m), 0.51–0.60 (2H,
m), 1.13–1.26 (1H, m), 1.30 (3H, d, J = 7.0 Hz), 1.82 (3H, s), 3.82 (2H,
d, J = 7.1 Hz), 4.85 (1H, quin, J = 7.2 Hz), 5.02 (2H, s), 6.65–6.73 (2H,
m), 6.76–6.85 (3H, m), 6.94–6.99 (2H, m), 7.23 (2H, d, J = 8.7 Hz), 7.34
(1H, t, J = 8.2 Hz), 8.20 (1H, d, J = 8.1 Hz). 13C NMR (75 MHz,
DMSO‑d6) δ 3.53 (2C), 10.49, 22.92, 23.14, 47.55, 57.92 (d, J = 9.75
Hz), 72.80, 101.87, 102.26, 106.86, 107.40 (t, J = 20.25 Hz), 111.96,
112.14, 114.81 (2C), 127.66 (2C), 131.28, 137.96, 156.13, 157.31,
159.78 (t, J = 14.25 Hz), 160.57, 160.71, 163.94 (d, J = 11.25 Hz),
168.54. LCMS m/z calcd for C27H27F2NO4: 467.19, found 468.1 [M + H]
+ .
N-{1-[6-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-difluor-
ophenyl}methoxy)pyridin-3-yl]ethyl}acetamide (1n): To a solution
of 29a (530 mg, 1.1 mmol) in THF (10 mL) was added MeMgBr (3 M
Et2O solution, 2.7 mL, 8.1 mmol) at 0 ◦C and the mixture was stirred at 0
◦C for 1 h. The mixture was quenched with sat. aq. NH4Cl and the
mixture was extracted with AcOEt. The organic layer was washed with
sat. aq. NaCl, dried over Na2SO4 and concentrated under reduced
pressure to afford the ketone. To a solution of the ketone in MeOH (15
mL) were added NH4OAc (500 mg, 6.5 mmol) and NaBH3CN (82 mg,
5.1.9. N-{1-[6-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-
difluorophenyl}methoxy)pyridin-3-yl]ethyl}acetamide (1n)
◦
1.3 mmol) at 30 C and the mixture was stirred at reflux for 12 h to
Methyl
6-[(2,6-difluoro-4-iodophenyl)methoxy]pyridine-3-
afford the amine. To the reaction mixture of the amine was added Ac2O
(204 mg, 2 mmol) at 30 ◦C and continued to stir at 30 ◦C for 2 h. The
mixture was concentrated under reduced pressure. The residue was
diluted with water and the mixture was extracted with AcOEt. The
organic layer was washed with sat. aq. NaCl, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by prep-
HPLC (0.1% NH3⋅H2O as additive) to afford 1n (137 mg, 45%) as a white
solid.
carboxylate (27a): To a solution of 24 (2 g, 7.4 mmol) in THF (20 mL)
was added NaH (60% in mineral oil, 296 mg, 7.4 mmol) portionwise at
30 ◦C and the mixture was stirred at 30 ◦C for 0.5 h. To the mixture was
added methyl 6-fluoropyridine-3-carboxylate (26a) (1.4 g, 8.9 mmol)
and the mixture was stirred at 30 ◦C for 2 h. The mixture was diluted
with water and extracted with AcOEt. The organic layer was washed
with sat. aq. NaCl, dried over Na2SO4 and concentrated under reduced
pressure. The residue was purified by column chromatography (silica
gel, eluent: 7/93 AcOEt/Petroleum ether) to afford a crude off-white
solid. The solid was triturated with AcOEt/Petroleum ether (2/1) to
afford 24 (1.1 g, 36%) as a white solid.
1H NMR (400 MHz, DMSO‑d6) δ 0.22–0.45 (2H, m), 0.47–0.60 (2H,
m), 1.10–1.30 (1H, m), 1.33 (3H, d, J = 6.8 Hz), 1.82 (3H, s), 3.81 (2H,
d, J = 7.2 Hz), 4.80–4.95 (1H, m), 5.28 (2H, s), 6.62–6.74 (2H, m),
6.75–6.85 (4H, m), 7.33 (1H, t, J = 8.4 Hz), 7.64 (1H, dd, J = 8.4, 2.4
Hz), 8.10 (1H, d, J = 2.0 Hz), 8.31 (1H, d, J = 7.6 Hz). 13C NMR (75
MHz, DMSO‑d6) δ 3.53 (2C), 10.48, 22.47, 23.07, 45.71, 55.24, 72.78,
101.86, 102.25, 106.81, 107.67 (t, J = 19.5 Hz), 110.69, 111.90,
112.10, 131.26, 134.11, 137.98, 144.78, 156.19, 159.62 (t, J = 14.25
Hz), 160.64, 160.70, 162.01, 164.01 (d, J = 10.5 Hz), 168.78. LCMS m/z
calcd for C26H26F2N2O4: 468.19, found 469.0 [M + H] + .
1H NMR (400 MHz, CDCl3) δ 3.91 (3H, s), 5.45 (2H, s), 6.76 (1H, d, J
= 8.4 Hz), 7.32 (2H, d, J = 6.4 Hz), 8.16 (1H, dd, J = 8.8, 2.4 Hz), 8.85
(1H, d, J = 2.4 Hz). LCMS m/z calcd for C14H10F2INO3: 404.97, found
405.9 [M + H] + .
Methyl
6-({4-[3-(cyclopropylmethoxy)phenoxy]-2,6-difluor-
ophenyl}methoxy)pyridine-3-carboxylate (28a): To a solution of
27a (870 mg, 2.2 mmol) in DMSO (15 mL) were added 10 (423 mg, 2.6
mmol), K3PO4 (912 mg, 4.3 mmol), CuI (99 mg, 0.52 mmol) and pico-
linic acid (127 mg, 1 mmol) at 30 ◦C and the mixture was stirred at 90 ◦C
under N2 atmosphere for 24 h. The mixture was cooled to room tem-
perature and diluted with water. The mixture was extracted with AcOEt.
The organic layer was washed with sat. aq. NaCl, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by col-
umn chromatography (silica gel, eluent: 7/93 AcOEt/Petroleum ether)
to afford 28a (600 mg, 63%) as a white solid.
5.1.10. N-{1-[5-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-
difluorophenyl}methoxy)pyridin-2-yl]ethyl}acetamide (1o)
Methyl
5-[(2,6-difluoro-4-iodophenyl)methoxy]pyridine-2-
carboxylate (27b): To a solution of 24 (3 g, 11 mmol) in THF (30 mL)
was added NaH (60% in mineral oil, 444 mg, 11 mmol) portionwise at
30 ◦C and the mixture was stirred at 30 ◦C for 0.5 h. To the mixture was
added
methyl
5-[(2,6-difluoro-4-iodophenyl)methoxy]pyridine-2-
carboxylate (26b) (2.1 g, 13 mmol) and the mixture was stirred at 30
◦C for 2 h. The mixture was diluted with water and extracted with
AcOEt. The organic layer was washed with sat. aq. NaCl, dried over
Na2SO4 and concentrated under reduced pressure. The residue was pu-
rified by column chromatography (silica gel, eluent: 33/67 AcOEt/Pe-
troleum ether) to afford 27b (2.4 g, 53%) as a white solid.
1H NMR (400 MHz, CDCl3) δ 0.25–0.40 (2H, m), 0.55–0.73 (2H, m),
1.15–1.35 (1H, m), 3.78 (2H, d, J = 7.2 Hz), 3.91 (3H, s), 5.43 (2H, s),
6.50–6.70 (4H, m), 6.71–6.80 (2H, m), 7.28 (1H, t, J = 8.0 Hz), 8.16
(1H, dd, J = 8.8, 2.4 Hz), 8.86 (1H, d, J = 2.0 Hz). LCMS m/z calcd for
C
24H21F2NO5: 441.14, found 442.1 [M + H] + .
6-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-difluorophenyl}
1H NMR (400 MHz, CDCl3) δ 3.98 (3H, s), 5.17 (2H, s), 7.30–7.45
(3H, m), 8.12 (1H, d, J = 8.8 Hz), 8.43 (1H, d, J = 2.8 Hz). LCMS m/z
calcd for C14H10F2INO3: 404.97, found 405.9 [M + H] + .
methoxy)-N-methoxy-N-methylpyridine-3-carboxamide (29a): To a
solution of 28a (600 mg, 1.4 mmol) in THF (6 mL) and MeOH (6 mL)
11

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
N-{1-[5-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-difluor-
mmol) in THF (10 mL) was added NaH (60% in mineral oil, 65 mg, 1.6
mmol) and the mixture was stirred at 15 ◦C for 30 min. To the mixture
was added 3,6-dibromopyridazine (31) (388 mg, 1.6 mmol), and the
mixture was stirred at 18 ◦C for 4 h. The reaction mixture was poured
into water and the mixture was extracted with AcOEt. The organic layer
was washed with sat. aq. NaCl, dried over Na2SO4 and concentrated
under reduced pressure. The residue was purified by column chroma-
tography (silica gel, eluent: 9/91 AcOEt/Petroleum ether) to afford 32
(570 mg, 75%) as a colorless oil.
ophenyl}methoxy)pyridin-2-yl]ethyl}acetamide (1o): To a solution
of compound 27b (1.0 g, 2.5 mmol) in DMSO (20 mL) was added
compound 2a (485 mg, 3 mmol), K3PO4 (1.1 g, 5 mmol), CuI (113 mg,
0.6 mmol) and picolinic acid (146 mg, 1.2 mmol) at 30 ◦C and the
mixture was stirred at 100 ◦C for 12 h. The mixture was cooled to room
temperature and diluted with water. The mixture was extracted with
AcOEt. The organic layer was washed with sat. aq. NaCl, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by column chromatography (silica gel, eluent: 33/67 AcOEt/
Petroleum ether) to afford the ester. To a solution of the eater in THF (6
mL) and MeOH (6 mL) were added LiOH⋅H2O (200 mg, 8.3 mmol) and
1H NMR (400 MHz, DMSO‑d6) δ 0.25–0.35 (2H, m), 0.50–0.60 (2H,
m), 1.15–1.25 (1H, m), 3.81 (2H, d, J = 7.2 Hz), 5.47 (2H, s), 6.65–6.73
(2H, m), 6.75–6.85 (3H, m), 7.28 (1H, d, J = 9.2 Hz), 7.34 (1H, t, J = 8.0
Hz), 7.91 (1H, d, J = 9.2 Hz). LCMS m/z calcd for C21H17BrF2N2O3:
462.04, found 462.9 [M + H] + .
◦
◦
water (6 mL) at 30 C. The mixture was stirred at 30 C for 2 h. The
mixture was concentrated under reduced pressure. The residue was
diluted with water and the mixture was acidified with 1 N aqueous HCl
and extracted with AcOEt. The organic layer was washed with sat. aq.
NaCl, dried over Na2SO4, and concentrated under reduced pressure to
afford the carboxylic acid. To a solution of the carboxylic acid in DMF
(10 mL) were added N,O-dimethylhydroxylamine hydrochloride (250
mg, 3.4 mmol), HOBt (568 mg, 4.2 mmol), EDCI (807 mg, 4.2 mmol)
and Et3N (850 mg, 8.4 mmol) at 30 ◦C and the mixture was stirred at 30
◦C for 3 h. The mixture was diluted with water and extracted with
AcOEt. The organic layer was washed with sat. aq. NaCl, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by column chromatography (silica gel, eluent: 33/67 AcOEt/
Petroleum ether) to afford the Weinreb amide. To a solution of the
Weinreb amide in THF (10 mL) was added MeMgBr (3 M Et2O solution,
2.7 mL, 8.1 mmol) dropwise at 0 ◦C and the mixture was stirred at 0 ◦C
for 1 h. The mixture was quenched with sat. aq. NH4Cl and extracted
with AcOEt. The organic layer was washed with sat. aq. NaCl, dried over
Na2SO4, and concentrated under reduced pressure to afford the ketone.
To a solution of the ketone in MeOH (15 mL) were added NH4OAc (550
mg, 7.1 mmol) and NaBH3CN (90 mg, 1.4 mmol) at 30 ◦C and the
mixture was stirred at reflux for 12 h to afford the amine. To the reaction
mixture of the amine was added Ac2O (204 mg, 2 mmol) at 30 ◦C and the
mixture was stirred at 30 ◦C for 1 h. The mixture was concentrated under
reduced pressure. The residue was diluted with water and extracted with
AcOEt. The organic layer was washed with sat. aq. NaCl, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by prep-HPLC (0.1% NH3⋅H2O as additive) to afford 1o (69 mg,
9%) as a white solid.
1-[6-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-difluor-
ophenyl}methoxy)pyridazin-3-yl]ethan-1-one (33): A mixture of 32
(570 mg, 1.2 mmol), tributyl(1-ethoxyvinyl)tin (535 mg, 1.5 mmol) and
Pd(PPh3)2Cl2 (18 mg, 0.025 mmol) in DMF (15 mL) was stirred at 80 ◦C
for 15 h under N2 atmosphere. To the mixture was added sat. aq. KF (40
mL) and the mixture was stirred at 20 ◦C for 1 h. The reaction mixture
was poured into water and the mixture was extracted with AcOEt. The
organic layer was washed with sat. aq. NaCl, dried over Na2SO4 and
concentrated under reduced pressure to afford a crude yellow solid. The
solid was dissolved in a mixture of 1 N HCl (20 mL) and acetone (20 mL),
and the mixture was stirred at 20 ◦C for 1 h. The mixture was neutralized
with sat. aq. NaHCO3 and extracted with AcOEt. The organic layer was
washed with sat. aq. NaCl, dried over Na2SO4 and concentrated under
reduced pressure. The residue was purified by column chromatography
(silica gel, eluent: 5/95 AcOEt/Petroleum ether) to afford 33 (300 mg,
57%) as a yellow solid.
1H NMR (400 MHz, DMSO‑d6) δ 0.25–0.35 (2H, m), 0.50–0.60 (2H,
m), 1.15–1.25 (1H, m), 2.72 (3H, s), 3.81 (2H, d, J = 6.8 Hz), 5.63 (2H,
s), 6.65–6.73 (2H, m), 6.75–6.85 (3H, m), 7.30–7.41 (2H, m), 8.05 (1H,
d, J = 8.8 Hz). LCMS m/z calcd for C23H20F2N2O4: 426.14, found 427.1
[M + H] + .
1-[6-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-difluor-
ophenyl}methoxy)pyridazin-3-yl]ethan-1-ol (34): To a mixture of
33 (300 mg, 0.7 mmol) in MeOH (10 mL) was added NaBH4 (53 mg, 1.4
mmol), and the mixture was stirred at 20 ◦C for 0.5 h. The reaction
mixture was poured into water and the mixture was extracted with
AcOEt. The organic layer was washed with sat. aq. NaCl, dried over
Na2SO4 and concentrated under reduced pressure to 34 (280 mg, 93%)
as a colorless oil.
1H NMR (400 MHz, DMSO‑d6) δ 0.20–0.36 (2H, m), 0.45–0.60 (2H,
m), 1.10–1.30 (1H, m), 1.32 (3H, d, J = 6.8 Hz), 1.84 (3H, s), 3.80 (2H,
d, J = 6.8 Hz), 4.80–4.99 (1H, m), 5.11 (2H, s), 6.65–6.71 (2H, m),
6.72–6.89 (3H, m), 7.25–7.40 (2H, m), 7.49 (1H, dd, J = 8.4, 2.8 Hz),
8.27 (1H, d, J = 2.8 Hz), 8.32 (1H, d, J = 8.0 Hz). LCMS m/z calcd for
1H NMR (400 MHz, DMSO‑d6) δ 0.25–0.35 (2H, m), 0.50–0.60 (2H,
m), 1.15–1.25 (1H, m), 1.40 (3H, d, J = 6.8 Hz), 3.82 (2H, d, J = 7.2 Hz),
4.85–4.95 (1H, m), 5.48 (2H, s), 5.55 (1H, d, J = 4.8 Hz), 6.65–6.73 (2H,
m), 6.75–6.88 (3H, m), 7.24 (1H, d, J = 9.2 Hz), 7.34 (1H, t, J = 8.0 Hz),
7.70 (1H, d, J = 8.8 Hz). LCMS m/z calcd for C23H22F2N2O4: 428.15,
found 429.0 [M + H] + .
C
26H26F2N2O4: 468.19, found 469.0 [M + H] + .
5.1.11. N-{1-[6-({4-[3-(Ccyclopropylmethoxy)phenoxy]-2,6-
difluorophenyl}methoxy)pyridazin-3-yl]ethyl}acetamide (1p)
3-(1-Azidoethyl)-6-({4-[3-(cyclopropylmethoxy)phenoxy]-2,6-
difluorophenyl}methoxy)pyridazine (35): To a mixture of 34 (280
mg, 0.65 mmol) and Et3N (198 mg, 2 mmol) in CH2Cl2 (10 mL) was
added MsCl (150 mg, 1.3 mmol) at 0 ◦C, and the mixture was stirred at
20 ◦C for 1 h. The reaction mixture was poured into water and extracted
with CH2Cl2. The organic layer was washed with water, dried over
Na2SO4 and concentrated under reduced pressure to afford the mesylate.
To a mixture of the mesylate in DMF (10 mL) was added NaN3 (213 mg,
3.3 mmol), and the mixture was stirred at 60 ◦C for 2 h. The reaction
mixture was poured into water and extracted with AcOEt. The organic
layer was washed with sat. aq. NaCl, dried over Na2SO4 and concen-
trated under reduced pressure. The residue was purified by column
chromatography (silica gel, eluent: 33/67 AcOEt/Petroleum ether) to
afford 35 (230 mg, 77%) as a colorless oil.
{4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-difluorophenyl}
methanol (30): A mixture of 24 (6 g, 22 mmol), 10 (3.7 g, 22 mmol),
CuI (423 mg, 2.2 mmol), picolinic acid (546 mg, 4.4 mmol) and K3PO4
(9.4 g, 44 mmol) in DMSO (60 mL) was stirred at 90 ◦C for 15 h under N2
atmosphere. The reaction mixture was poured into water and the
mixture was extracted with AcOEt. The organic layer was washed with
sat. aq. NaCl, dried over Na2SO4 and concentrated under reduced
pressure. The residue was purified by column chromatography (silica
gel, eluent: 9/91 AcOEt/Petroleum ether) to afford 30 (3.7 g, 54%) as a
yellow oil.
1H NMR (400 MHz, DMSO‑d6) δ 0.25–0.35 (2H, m), 0.50–0.60 (2H,
m), 1.15–1.25 (1H, m), 3.80 (2H, d, J = 6.8 Hz), 4.44 (2H, d, J = 5.6 Hz),
5.20 (1H, t, J = 5.6 Hz), 6.60–6.68 (2H, m), 6.71 (2H, d, J = 8.8 Hz),
6.80 (1H, d, J = 7.6 Hz), 7.32 (1H, t, J = 8.0 Hz).
1H NMR (400 MHz, DMSO‑d6) δ 0.25–0.35 (2H, m), 0.50–0.60 (2H,
m), 1.15–1.25 (1H, m), 1.57 (3H, d, J = 6.8 Hz), 3.81 (2H, d, J = 7.2 Hz),
4.94–5.00 (1H, m), 5.51 (2H, s), 6.65–6.73 (2H, m), 6.78–6.88 (3H, m),
3-Bromo-6-({4-[3-(cyclopropylmethoxy)phenoxy]-2,6-difluor-
ophenyl}methoxy)pyridazine (32): A mixture of 30 (500 mg, 1.6
12

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
7.30–7.29 (2H, m), 7.74 (1H, d, J = 9.2 Hz). LCMS m/z calcd for
complete protease inhibitor), and subjected to ultracentrifuge at
185700xg for 50 min at 4 ◦C. ACC protein with 6xHis-tag at the N-ter-
minal was purified from the supernatant using Ni-NTA Super Flow Gel
(QIAGEN). Eluted protein was dialyzed against buffer B (50 mM HEPES
(pH7.5), 300 mM NaCl, 10 mM MgCl2, 10 mM tripotassium citrate, 2
mM dithiothreitol) and concentrated using Amicon Ultra (Merck).
Compounds were dissolved in DMSO and then diluted with an enzyme
reaction buffer (50 mM HEPES (pH 7.5), 10 mM MgCl2, 10 mM tri-
pottasium citrate, 2 mM dithiothreitol, 0.001% fatty acid-free BSA).
Recombinant human ACC1 or ACC2 was diluted with the enzyme re-
C
23H21F2N5O3: 453.16, found 454.0 [M + H] + .
N-{1-[6-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-difluor-
ophenyl}methoxy)pyridazin-3-yl]ethyl}acetamide (1p): A mixture
of 35 (230 mg, 0.51 mmol) and Ph3P (200 mg, 0.76 mmol) in THF (10
mL) and water (1 mL) was refluxed for 15 h. To the reaction mixture was
added Ac2O (104 mg, 1 mmol), and the mixture was stirred at 20 ◦C for
1 h. The reaction mixture was poured into water and extracted with
AcOEt. The organic layer was washed with sat. aq. NaCl, dried over
Na2SO4 and concentrated under reduced pressure. The residue was pu-
rified by prep-HPLC (0.1% NH4HCO3 as additive) to afford 1p (123 mg,
52%) as a white solid.
action buffer to a concentration of 0.2
pound solution was added to each well of a 384-well assay plate, and 5
L of the enzyme mixture was added to each well. The mixture was
incubated at room temperature for 60 min. Then, a substrate solution
(50 mM KHCO3, 200 M ATP, 200 M acetyl-CoA, 5 L) was added to
each well, and the mixture was reacted at room temperature for 30 min.
μg/mL. A 5 μL aliquot of com-
1H NMR (400 MHz, DMSO‑d6) δ 0.25–0.35 (2H, m), 0.50–0.60 (2H,
m), 1.15–1.25 (1H, m), 1.41 (3H, d, J = 7.2 Hz), 1.85 (3H, s), 3.81 (2H,
d, J = 6.8 Hz), 5.02–5.11 (1H, m), 5.48 (2H, s), 6.65–6.73 (2H, m),
6.75–6.86 (3H, m), 7.21 (1H, d, J = 9.2 Hz), 7.34 (1H, t, J = 8.0 Hz),
7.56 (1H, d, J = 8.8 Hz), 8.42 (1H, d, J = 8.0 Hz). 13C NMR (75 MHz,
DMSO‑d6) δ 3.53 (2C), 10.48, 21.07, 23.00, 48.50, 56.55, 72.79, 101.89,
102.27, 106.85, 107.13 (t, J = 20.25 Hz), 111.95, 112.14, 118.06,
128.96, 131.29, 156.13, 159.89 (t, J = 15 Hz), 160.71, 160.82, 161.13,
163.86, 164.04 (d, J = 10.5 Hz), 169.23. LCMS m/z calcd for
μ
μ
μ
μ
The reaction was stopped by adding a 40 μL of stop solution (1.3%
formic acid, 0.2
μ
M malonyl-13C3-CoA) to each of the obtained reaction
mixtures. The production of malonyl-CoA was detected by RapidFire-
Mass spectrometry and corrected by malonyl-13C3-CoA. IC50 values
were calculated by XLfit from the data expressed as inhibition (%) using
fit Model 204 (4 Parameter Logistic Model). The response of vehicle
control was set as 0% inhibition and the response without enzyme was
set as 100% inhibition.
C
25H25F2N3O4: 469.18, found 470.0 [M + H] + .
5.1.12. N-{1-[5-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-
difluorophenyl}methoxy)pyrazin-2-yl]ethyl}acetamide (1q)
1-[5-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-difluor-
ophenyl}methoxy)pyrazin-2-yl]ethan-1-one (37): A mixture of 30
(60 mg, 0.2 mmol), 1-(5-chloropyrazin-2-yl)ethan-1-one (36) (34 mg,
0.22 mmol) and NaH (60% in mineral oil, 12 mg, 0.29 mmol) in THF (1
mL) was stirred at room temperature for 10 min. The mixture was heated
to 135 ◦C for 1 h under microwave irradiation. The mixture was
5.3. Acetate uptake assay
HCT-116 cells (ATCC) were plated in a 96-well cell culture plate at
50,000 cells/well and incubated overnight in RPMI medium, supple-
mented with 10% fetal bovine serum, penicillin (10,000 unit/mL), and
streptomycin (10,000
washed twice with 100
μ
g/mL), under 5% CO2 at 37 ◦C. The cells were
◦
quenched with water at 0 C and extracted with AcOEt. The organic
μ
L PBS and incubated with 90 μL of test com-
layer was separated, washed with sat. aq. NaCl, dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by prep-
HPLC (0.1% TFA additive) to afford 37 (20 mg, 24%).
pounds in assay medium (RPMI and 0.1% fatty acid-free BSA) for 60
min. Then, 0.1
μ
Ci/well of [14C]acetic acid was added to the well and
incubated for 2 h at 37 ◦C. Cells were washed twice with 100
μL of PBS to
1H NMR (300 MHz, CDCl3) δ 0.31–0.42 (2H, m), 0.61–0.70 (2H, m),
1.22–1.30 (1H, m), 2.66 (3H, s), 3.79 (2H, d, J = 7.0 Hz), 5.47 (2H, s),
6.53–6.68 (4H, m), 6.76 (1H, ddd, J = 8.3, 2.4, 0.8 Hz), 7.27–7.34 (1H,
m), 8.18–8.23 (1H, m), 8.81–8.88 (1H, m). LCMS m/z calcd for
remove the radioactive medium, and 60 μL of Microscint20 was added.
The radioactivity in each well was measured by Topcount (Perki-
nElmer). The well containing 100 nM reference compound (compound
1f in ref. 34) was used as a 100% inhibition control. The well containing
DMSO was used as a 0% inhibition control. IC50 values were calculated
by XLfit from the data expressed as inhibition (%) using fit Model 204 (4
Parameter Logistic Model).
C
23H20F2N2O4: 426.14, found 427.2 [M + H] + .
N-{1-[5-({4-[3-(Cyclopropylmethoxy)phenoxy]-2,6-difluor-
ophenyl}methoxy)pyrazin-2-yl]ethyl}acetamide (1q): A mixture of
37 (12 mg, 0.03 mmol), NaBH3CN (3.5 mg, 0.06 mmol) and NH4OAc
(11 mg, 0.14 mmol) in MeOH (1 mL) was stirred at 65 ◦C overnight. The
mixture was quenched with water at room temperature and extracted
with AcOEt. The organic layer was separated, washed with sat. aq. NaCl,
dried over MgSO4 and concentrated under reduced pressure to afford the
amine. To a mixture of the amine in THF (1 mL) was added Ac2O (0.008
mL, 0.08 mmol) at room temperature. The mixture was stirred at room
temperature for 1 h. The mixture was quenched with water at room
temperature and extracted with AcOEt. The organic layer was separated,
washed with sat. aq. NaCl, dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by prep-HPLC (0.1% TFA
additive) to afford 1q (7 mg, 53%) as white amorphous powder.
1H NMR (300 MHz, CD3OD) δ 0.30–0.40 (2H, m), 0.56–0.67 (2H, m),
1.18–1.26 (1H, m), 1.47 (3H, d, J = 7.2 Hz), 1.97 (3H, s), 3.81 (2H, d, J
= 6.8 Hz), 5.02–5.11 (1H, m), 5.40 (2H, s), 6.53–6.68 (4H, m),
6.77–6.85 (1H, m), 7.24–7.36 (1H, m), 8.14 (2H, s). LCMS m/z calcd for
5.4. In vivo PD study
All experimental procedures were approved by the Institutional
Animal Care and Use Committee of Takeda Pharmaceutical Company
Ltd., which is fully accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care International. Athymic nude
mice (female BALB/cA Jcl-nu/nu) and HCT-116 human colorectal car-
cinoma cell line were purchased from CLEA (Tokyo, Japan) and ATCC
(American Type Culture Collection), respectively. Mice (6 weeks old)
received s.c. injections into the hind flank with 100 μL cultured HCT-116
cancer cell suspension in Hanks’ balanced salt solution (Invitrogen) with
Matrigel (BD Cat.356237). After the tumor xenografts were established,
the animals were randomly grouped by using tumor volume. Tumor
volumes were assessed by measurement of two dimensions with vernier
calipers and were calculated as length × width2 × 1/2. The mice with s.
c. HCT-116 xenografts were orally given the vehicle (0.5% methyl cel-
lulose, Wako) or compound 1n at 100 mg/kg. Whole blood and tumor
tissue samples were collected after 2 and 16 h treatment. The blood was
centrifuged to collect plasma, and tumor tissues were snap frozen for
measurement of malonyl-CoA. Concentration of malonyl-CoA in tumor
was measured by reverse-phase liquid chromatography-tandem mass
spectrometry (RPLC-MS/MS). The frozen samples were pulverized
under liquid nitrogen. The powdered tissue was mixed with [13C3]-
C
25H25F2N3O4: 469.18, found 470.0 [M + H] + .
5.2. ACC enzyme assay
SF-9 cells were infected with the baculovirus containing human or
mouse ACC and cultured at 27 ◦C for 3 days. Harvested cells were ho-
mogenized in buffer A (50 mM HEPES (pH7.5), 300 mM NaCl, 25 mM
sodium glycerophosphate, 1 mM sodium orthovanadate, 10% glycerol,
13

R. Mizojiri et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116056
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malonyl-CoA, and then homogenized with 6% aqueous perchloric acid.
After centrifugation, the supernatants were applied onto an Oasis HLB
cartridge (Waters, Milford, MA) for solid-phase extraction. The sample-
loaded cartridges were washed with water and then eluted with aceto-
nitrile/water/dibutylammonium acetate (500:500:1, v/v/v). The eluted
samples were centrifuged, and the supernatants were used for RPLC-MS/
MS. For the liquid chromatography separation, the samples were
injected to a Capcell PAK C18 AQ (Shiseido, Tokyo, Japan) with column
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◦
temperature at 40 C. Chromatographic separation was performed by
gradient elution of two mobile phases: mobile phase A consisted of 5
mmol/L ammonium acetate/dibutylammonium acetate (100:1, v/v, pH
9.0), and mobile phase B consisted of acetonitrile. Malonyl-CoA and
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[
13C3]-malonyl-CoA were detected using a mass spectrometer by mul-
tiple reaction monitoring (MRM) with transitions of m/z 852.0 → 808.0
for malonyl-CoA and m/z 855.0 → 810.0 for [13C3]–malonyl-CoA,
respectively. The malonyl-CoA concentration was determined using a
calibration curve, which was calculated by the responses of given con-
centrations of standard reagents normalized by the response of [13C3]–
malonyl-CoA as an internal standard.
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Data were represented as mean ± standard deviation (SD). Statistical
differences between the control and the treated groups were analyzed by
Dunnett’s test, and a value of P < 0.05 was considered significant.
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Mammalian Acetyl CoA-carboxylase. Bioorg Med. Chem. Lett. 2015;25:5352–5356.
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Author contributions
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript. R.
M., D. T., and H. M. contributed design and synthesis of compounds; M.
S., Y. S., Y. Y., and H. S. contributed in vitro and in vivo study.
22 Kamata M, Yamashita T, Kina A, et al. Symmetrical Approach of Spiro-
pyrazolidinediones as Acetyl-CoA Carboxylase Inhibitors. Bioorg Med. Chem. Lett.
2012;22:4769–4772.
23 Kamata M, Yamashita T, Kina A, et al. Design, Synthesis, and Structure-activity
Relationships of Novel Spiro-piperidines as Acetyl-CoA Carboxylase Inhibitors.
Bioorg Med. Chem. Lett. 2012;22:3643–3647.
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgements
24 Chonan T, Wakasugi D, Yamamoto D, et al. Discovery of Novel (4-Piperidinyl)-
piperazines as Potent and Orally Active Acetyl-CoA Carboxylase 1/2 Non-selective
Inhibitors: F-Boc and triF-Boc Groups are Acid-stable Bioisosteres for the Boc Group.
Bioorg Med Chem. 2011;19:1580–1593.
25 Keil S, Müller M, Zoller G, et al. Identification and Synthesis of Novel Inhibitors of
Acetyl-CoA Carboxylase with In Vitro and In Vivo Efficacy on Fat Oxidation. J Med
Chem. 2010;53:8679–8687.
26 Corbett, J. W.; Freeman-Cook, K. D.; Elliott, R.; Vajdos, F,; Rajamohan, F.; Kohls, D.;
Marr, E.; Zhang, H.; Tong, L.; Tu, M.; Murdande, S.; Doran, S. D.; Houser, J. A.; Song,
W.; Jones, C. J.; Coffey, S. B.; Buzon, L.; Minich, M. L.; Dirico, K. J.; Tapley, S.;
McPherson, R. K.; Sugarman, E.; Harwood, H.J. Jr.; Esler, W. Discovery of Small
Molecule Isozyme Non-specific Inhibitors of Mammalian Acetyl-CoA Carboxylase 1
and 2 Bioorg. Med. Chem. Lett. 2010, 20, 2383–2388.
We sincerely appreciate Drs. Hiroshi Miyake, Takahito Hara,
Tomoyasu Ishikawa, Yuichi Hikichi, for intellectual support during this
work. We also appreciate Drs. Yasuhiro Imaeda and HugoKaHo Fong, for
helpful suggestions during the preparation of this manuscript. We would
like to express our appreciation to Moriteru Asano, Hiroshi Banno,
Noriyuki Nii and Krista E. Gipson for derivative synthesis; Takeo Moriya
and Yoshinori Satomi for evaluating in vivo PD test; Kazunobu Aoyama
and Naomi Kamiguchi for supporting in PK studies; Yoshitaka Inui for
helping in drug safety; Youko Ishihara for contributing formulation
analyses.
27 Haque TS, Liang N, Golla R, et al. Potent Biphenyl- and 3-Phenyl Pyridine-based
Inhibitors of Acetyl-CoA Carboxylase. Bioorg Med. Chem. Lett. 2009;19:5872–5876.
28 Wu X, Huang T. Recent Development in Acetyl-CoA Carboxylase Inhibitors and Their
Potential as Novel Drugs. Future Med Chem. 2020;12:533–561.
29 Chen L, Duan Y, Wei H, et al. Acetyl-CoA Carboxylase (ACC) as a Therapeutic Target
for Metabolic Syndrome and Recent Developments in ACC1/2 inhibitors. Expert Opin
Investig Drugs. 2019;28:917–930.
30 Corbett JW. Review of Recent Acetyl-CoA Carboxylase Inhibitor Patents: Mid-2007 –
2008. Expert Opin Ther Patents. 2009;19:943–956.
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