Syntheses and pharmacokinetic evaluations of four metabolites of 2-(4-(2-((1H-benzo[d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis-(methylthio)pyridin-3-yl)acetamide hydrochloride [K-604], an acyl-CoA:cholesterol O-acyltransferase-1 inhibi

Article abstract of DOI:10.1016/j.bmc.2020.115457

We synthesized and identified four metabolites of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT)-1 inhibitor, K-604 (1). Two of the metabolites M1 and M2, were prepared from 1 using a combination reagent of hydrogen peroxide and sodium tungstate wit

Full text of DOI:10.1016/j.bmc.2020.115457

Journal Pre-proofs
Syntheses and pharmacokinetic evaluations of four metabolites of 2-(4-(2-
((1H-benzo[d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis-
(methylthio)pyridin-3-yl)acetamide hydrochloride [K-604], an acyl-CoA:cho‐
lesterol O-acyltransferase-1 inhibitor
Kimiyuki Shibuya, Toru Miura, Tadaaki Ohgiya, Kozo Omichi, Yoshihiko
Tsunenari
PII:
DOI:
Reference:
S0968-0896(20)30271-6
https://doi.org/10.1016/j.bmc.2020.115457
BMC 115457
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
15 January 2020
17 March 2020
20 March 2020
Please cite this article as: Shibuya, K., Miura, T., Ohgiya, T., Omichi, K., Tsunenari, Y., Syntheses and
pharmacokinetic evaluations of four metabolites of 2-(4-(2-((1H-benzo[d]imidazol-2-yl)thio)ethyl)piperazin-1-
yl)-N-(6-methyl-2,4-bis-(methylthio)pyridin-3-yl)acetamide hydrochloride [K-604], an acyl-CoA:cholesterol O-
acyltransferase-1 inhibitor, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.
2020.115457
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

Syntheses and pharmacokinetic evaluations of four
metabolites of 2-(4-(2-((1H-benzo[d]imidazol-2-
yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis-
(methylthio)pyridin-3-yl)acetamide hydrochloride
[K-604], an acyl-CoA:cholesterol O-acyltransferase-
1 inhibitor
Kimiyuki Shibuya,†, Toru Miura †, Tadaaki Ohgiya *†, Kozo Omichi ‡, Yoshihiko Tsunenari †.
†Tokyo New Drug Research Laboratories, Pharmaceutical Division, Kowa Company, Ltd., 2-
17-43, Noguchicho, Higashimurayama, Tokyo 189-0022, Japan
‡Fuji Research Laboratories, Pharmaceutical Division, Kowa Company, Ltd., 332-1,
Ohnoshinden, Fuji, Shizuoka 417-8650, Japan
KEYWORDS. acyl-CoA:cholesterol O-acyltransferase, ACAT-1 inhibitor, K-604, metabolite,
monosulfoxidation, pharmacokinetics, microsomes, clearance, CYP3A4, metabolic pattern.
1

ABSTRACT. We synthesized and identified four metabolites of acyl-coenzyme A:cholesterol O-
acyltransferase (ACAT)-1 inhibitor, K-604 (1). Two of the metabolites M1 and M2, were prepared
from 1 using a combination reagent of hydrogen peroxide and sodium tungstate with either
phosphoric acid or trifluoroethanol as the solvent to control the regioselectivity. Upon exposure of
4b to tert-butyl hypochlorite at -78 °C, the monosulfoxidation afforded synthetic intermediate of
M3 in excellent yield. The efficient synthesis of M4 was established. The in vitro metabolic study
exhibited a high clearance value (720 L/min/mg protein) of 1 using human liver microsomes. We
orally administered a single dose of 10 mg/kg of 1 to monkeys because the in vitro metabolic
patterns are quite similar. Fortunately, the drug concentration of 1 was much higher than those of
M1, M2, M3 and M4.
1. Introduction
Recently, we reported the discovery of clinical candidate 2-(4-(2-((1H-benzo[d]imidazol-2-
yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis-(methylthio)pyridin-3-yl)acetamide
hydrochloride (1, K-604), an acyl-coenzyme A:cholesterol O-acyltransferase (ACAT), also known
1-8
as a sterol O-acyltransferase (SOAT) -1 inhibitor.
Understanding the metabolism and
pharmacokinetics of drug candidates is crucial for reducing the high attrition rate in the
9-12
development of novel drugs.
However, detecting trace amounts of drug metabolites in each
species and determining their chemical structures can be quite difficult, as they are often mono-
oxygenated or monohydroxylated derivatives of the parent compound. 13 Furthermore, a challenge
for medicinal chemists is obtaining a sufficient amount of the metabolites for toxicity tests without
substantial process chemistry studies in the early stage of drug development. 14 The evaluation and
interpretation of the in vitro metabolism data and in vivo pharmacokinetic (PK) data are
occasionally used to make go/no-go decisions at the clinical step. In the present study, we disclose
2

a synthetic study on the preparation of a series of desired metabolites in an efficient manner and
the PK results of a parent drug 1 (K-604) and its metabolites, M1, M2, M3, and M4 through in
vitro and in vivo experiments.
2. Chemistry
Based on a simulation by StarDropTM, 15 we first proposed four metabolites, that could be derived
from the oxygenation or hydroxylation of the electron-rich positions of parent compound 1 (K-
604), as shown in Figure 1.
S
369
H
N
N
H
N
N
N
O
S
S
N
O
M2
150
O
S
S
S
S
369
353
S
S
H
H
N
N
H
N
H
N
N
H
N
N
N
N
N
N
O
H
N
N
O
N
S
S
N
O
N
S
N
HCl
N
O
M3
M1
150
166
1 (K-604)
S
S
353
H
N
N
H
N
N
N
O
S
N
HO
M4
166
Figure 1. Chemical structures of M1, M2, M3 and M4 metabolized from 1 (K-604).
To synthesize the metabolites M1 and M2, we first prepared common intermediates 4a and 4b via
3a and 3b starting from N-2-hydroxyethylpiperazine derivatives 2a and 2b, as shown in Scheme
1.
3

Scheme 1. Initial syntheses of M1 and M2.
O
O
NH
N
R
N
R
H
N
H
N
i
ii or iii
N
N
N
S
HO
S
N
N
2a
2b
3a
3b
4a
3 HCl salt
R = H
R = OC(CH3)3
R = H
R = OC(CH3)3
4b
3 CF3COOH salt
O
O
NOE
S
S
4
H
5
iv
v
H
N
6
H
N
3
N
Br
H
N
N
N
1
2
O
N
O
S
S
S
N
S
H
N
6
M1
Br
N
S
O
NOE
S
S
H
S
vi
vii
H
5
H
N
N
N
Br
H
N
N
N
N
O
O
S
S
N
O
O
7
M2
a Reagents and conditions: i) Et3N, DMAP, MsCl, THF, 0 °C, then 2-mercaptobenzimidazole,
K2CO3, 18-crown-6, DMF, 80 °C, 74% for 3a and 64% for 3b over 2 steps; ii) 12 N HCl,
MeOH, reflux, 97%; iii) CF3COOH, 0 °C to rt, 64%; iv) NaBO3, AcOH, 0 °C to rt, 6%; v) 4b,
K2CO3, H2O, DMF, rt, 75%; vi) m-CPBA, CHCl3, −20 °C; vii) 4b, K2CO3, MeCN, 0 °C to rt,
33% over 2 steps.
Following known methods, 2, 16, 17 both compounds, 1-formyl-4-(2-hydroxyethyl)piperazine (2a)
and 1-tert-butoxycarbonyl-4-(2-hydroxyethyl)piperazine (2b), were converted to their
corresponding thioethers (3a and 3b) via their mesylates under the reactions described above. 3a
and 3b were deprotected to afford corresponding compounds 4a and 4b under acidic conditions.
In our first attempt using conventional oxidation conditions, such as peracetic acid, the selective
monosulfoxidation of the bis(methylthio) groups of 5 resulted in a complicated mixture of products
4

containing trace amounts of desired compounds 6 and 7. After exploring other oxidation reagents,
we found that exposing intermediate 5 to sodium perborate 18 in AcOH afforded a small amount
1
of 6 (6% yield) after careful separation and purification. The H-NMR spectrum confirmed the
presence of a methylsulfinyl group at the 4-position of 6 because the irradiation of the hydrogen
atom (7.39 ppm) at the 5-position gave a positive nuclear Overhauser enhancement (NOE) of the
methylsulfinyl group (2.71 ppm) at the 4-position and the methyl group (2.49 ppm) at the 6-
position, which were consistent with the putative adjacent groups. Thus, 6 was reacted with 4b
under basic conditions to give M1 in 75% yield. Monosulfoxide 7 was obtained by treating 5 with
m-chloroperoxybenzoic acid (m-CPBA) at −20 °C, but it could not be isolated due to its instability.
Therefore, we used the crude mixture containing 7 without purification in the next step and
obtained desired compound M2 in 33% yield over 2 steps. The structure of M2 was also assigned
based on NOE. Although these synthetic methods are suitable for subsequent PK studies, we
needed to develop efficient methods for the large-scale preparation of M1 and M2 to allow long-
term in vivo toxicity tests.
Scheme 2. Facile syntheses of M1 and M2 via the direct oxidation of 1 for large-scale preparation.
O
S
S
S
S
H
H
i
ii
H
H
1
(K-604)
N
N
N
N
H
N
H
N
N
N
N
O
N
O
S
S
N
N
O
M1
M2
a Reagents and conditions: i) 30% H2O2, Na2WO4·2H2O, [CH3(n-C8H17)3N]HSO4, MeOH, rt, 47%;
ii) 30% H2O2, Na2WO4·2H2O, [CH3(n-C8H17)3N]HSO4, H2SO4/H3PO4, rt, 54%.
5

To avoid undesirable overoxidation and the synthetic route involving unstable intermediate 7, we
developed an alternative synthetic route for preparing M1 and M2 starting from a common parent
compound (1) via a catalytic oxidation, as shown in Scheme 2. We focused on the oxidation of the
sulfide to a sulfoxide using a catalytic oxidation system, such as a combination of sodium tungstate
dihydrate (Na2WO4·2H2O), methyltri(n-octyl)ammonium hydrogen sulfate ([CH3(n-
19-21
C8H17)3N]HSO4) and 30% hydrogen peroxide (H2O2).
After optimization of the reaction
conditions, no overoxidation of the sulfoxide into the sulfone was observed. We established a
regioselective method to obtain either M1 or M2 based on the selected solvent, as shown in Table
1.
Table 1. Proportion of M1 and M2 obtained from the tungsten oxidation of 1 in a variety of
solvents.
Entry
Solvent
MeOH
M1
83
82
61
92
86
35
32
34
M2
17
18
39
8
1
2
3
4
5
6
7
8
AcOH
CF3COOH
CF3CH2OH
(CF3)2CHOH
H2SO4
14
65
68
66
H3PO4
H2SO4/H3PO4
a Reaction was run using 30% H2O2, Na2WO4·2H2O, [CH3(n-C8H17)3N]HSO4 and compound 1
in a 1100-660:1:1:92.5 molar ratio at rt. The ratio between M1 and M2 was determined by HPLC
analysis.
6

Interestingly, compound M1 was selectively produced in the polar solvent of trifluoroethanol 22
(M1:M2 = 92:8, entry 4). In contrast, the proportion of M1 decreased and compound M2 was the
major product when the reaction was conducted in strong acids, such as sulfuric acid, phosphoric
acid and their mixture (M1:M2 = 32-35:68-65, entries 6, 7, 8). Considering these results, we
calculated the density of the electron charge on the bis(methylthio) groups in the model structures
of N-(6-methyl-2,4-bis(methylthio)pyridin-3-yl)acetamide (8) and 3-acetamido-6-methyl-2,4-
bis(methylthio)pyridin-1-ium (9) using DFT calculation at the theoretical level of B3LYP-D3 with
6-31G** basis set (Schrödinger Jaguar 10.2). 23 In the model structure of 8, the electron density (−
0.307) on the sufur atom at the 4-position with the methylthio group is higher than the density (−
0.219) on the sulfur at the 2-position with the methylthio group. Therefore, the oxidation
preferentially occurred at the 4-position (at the electron-rich sulfur atom) to provide oxidation
product M1 over M2. In the model structure of 9, the electron density (− 0.157) on the sulfur atom
at the 2-position with the methylthio group is higher than the electron density (− 0.024) on the
sulfur at the 4 position with the methylthio group, and as a result, M2 was generated as the major
product, as shown in Figure 2.
Oxidation
S
-0.219 S
-0.157
4
4
H
N
H
N
2
S
N
-0.090
2
N
-0.473
O
O
S
H
-0.024
0.269
-0.307
Oxidation
9
8
Figure 2. Comparison of the electron densities on the bis(methylthio) groups in 8 and 9.
For large-scale production, we carried out the reaction compound 1 (10-g scale) with a
combination of Na2WO4·2H2O, [CH3(n-C8H17)3N]HSO4 and 30% H2O2 in a mixture of conc.
7

H2SO4 and H3PO4 to afford M1 in 47% yield, and multiple batches were prepared. Similarly, we
performed the tungsten oxidation of 1 in MeOH to give M2 in 52% yield.
Scheme 3. Synthesis of M3
S
H
N
N
H
N
NH
H
N
N
i
ii
N
O
N
S
4b
S
S
N
N
O
O
10
M3
a Reagents and conditions: i) t-BuOCl, MeOH, −78 °C, 99%; ii) 5, K2CO3, MeCN, rt, 69%.
Next, we addressed the efficient preparation of sulfoxide M3. To protect the reactive nitrogen of
the piperazine moiety with tris(trifluoroacetate) during the oxidation of sulfur atom, we reacted
soluble trifluoroacetate salt 4b with tert-butyl hypochlorite (t-BuOCl) in MeOH at −78 °C. 24 The
sulfur oxidation cleanly afforded desired compound 10 in 99% yield. We then coupled 5 and 10 in
the presence of K2CO3 to give desired compound M3 in 69% yield.
Scheme 4. Synthesis of M4
8

H
N
NH2
NO2
NH2
NO2
i
NH2
NH2
iii
ii
SH
N
HO
BnO
BnO
BnO
12
14
11
13
S
S
S
S
iv
v
H
H
N
N
N
N
H
N
N
N
N
O
N
O
HO
S
N
BnO
16
15
S
S
H
N
N
H
N
N
N
O
S
N
HO
M4
a Reagents and conditions: i) BnBr, K2CO3, acetone, rt, 86%; ii) Zn dust, conc. HCl, AcOH, 0 °C
to rt; iii) KS(C=S)OEt, EtOH, reflux, 62% over 2 steps; iv) Et3N, DMAP, MsCl, THF, then 14,
K2CO3, 18-crown-6, DMF, rt to 80 °C, 88% over 2 steps; v) MeSPh, CF3COOH, 50 °C, 98%.
To elaborate the 5-hydroxy-2-mercaptobenzimidazole unit, we began by protecting 4-amino-3-
nitrophenol (11) with benzylbromide under basic conditions to give benzyl ether 12, which was
reduced with zinc dust in conc. HCl/AcOH to afford o-phenylenediamine 13. The cyclization of
13 proceeded upon treatment with potassium ethylxanthogenate in refluxing EtOH to yield
cyclized compound 14. The conventional coupling reaction between 14 and 15 furnished product
16 in high yield. Finally, we cleaved the benzyl protecting group from 16 by treatment with
thioanisole in CF3COOH via a push-pull mechanism 25 and obtained desired phenol M4 in 98%
yield.
3. Results and discussion
3.1. Determination of Four Metabolites M1, M2, M3 and M4, using Human Liver
Microsomes
9

To identify the structures of these four metabolites, we conducted metabolic tests of 1 using human
liver microsomes as follows. The structures of the four proposed metabolites, M1 (m/z 518.9; Rt
3.37 min), M2 (m/z 518.9; Rt 2.73 min), M3 (m/z 518.9; Rt 5.78 min), and M4 (m/z 518.9; Rt
3.21 min) were determined by LC/MS/MS analysis (Supporting Information). These all
metabolites indicated no ACAT inhibitory activities, whereas parent compound 1 showed potent
ACAT inhibition. 1
3.2. In vitro metabolism and kinetic analysis using human liver microsomes
To clarify the metabolism of 1, we kinetically analyzed the formation rate of the four metabolites,
M1, M2, M3 and M4, using human liver microsomes. The cytochrome P (CYP) isoforms involved
in the formation of these metabolites were identified using baculovirus expressed human CYP
microsomes. 26, 27 Metabolites M1, M2, M3 and M4 were formed enzymatically in the presence
of NADPH. The metabolic clearance (CL) of 1 was very high at 720 L/min/mg protein, which
suggested that parent compound 1 is likely to be metabolized. 28-30 The Km values (mean ± S. D.)
of M1, M2, M3 and M4 in the metabolism of 1 were 1.3 ± 0.9, 4.3 ± 3.9, 2.6 ± 0.1, and 2.0 ± 0.1
mol/L, respectively. The Vmax values (mean ± S. D.) of these metabolites were 1773 ± 18, 363
± 137, 558 ± 20, and 343 ± 12 pmol/min/mg protein, respectively. The CL (Vmax/Km) values
(mean ± S. D.) of M1, M2, M3 and M4 were 1353 ± 34, 125 ± 39, 219 ± 4, and 168 ± 12
L/min/mg protein, respectively, which suggested that M1 is the major metabolite in HLM
(human liver microsomes) test. CYP3A4 and 3A5 were the primary enzymes involved in the
formation of M1, M2, M3 and M4. CYP2C9 did not afford any metabolites. CYP2D6 was
involved only in the formation of M4 (Supporting Information).
10

3.3. Assessment of the similarities among human and other species’ metabolic patterns of 1
using the corresponding microsomes for in vitro experiment
To assess the similarities among the metabolic patterns of 1 in humans and other animal species,
we conducted HPLC-radioluminography using the corresponding microsomes for in vitro
experiments 31 and obtained significant differences, as shown in Figure 5. These results revealed
that human metabolic pattern resembled that of male mice pattern and was rather similar to that of
monkeys. Male rats produced many metabolites including amounts of M1 and M2 and a trace
amount of 1 (5.2%), whereas female rats primarily retained 1 (76.1%) and generated a small
amount of M1 (Supporting Information). This was due to gender differences in rat metabolism.
CYP3A2 is involved in metabolism in male rats, whereas CYP2C11 dominated metabolism in
female rats. Unexpectedly, we could not specifically identify M4 in in vitro studies. All species
produced M1 because it showed the highest CL value among those of the four metabolites from
1. Here, we wondered if parent drug 1 could be maintained at a sufficient level or would be rapidly
cleared in animal studies.
11

Figure 5. Comparison of the in vitro metabolic patterns of 1 (K-604) using microsomes from
humans and other animal species.
After a single oral administration of 10 mg/kg of 1 to fasted male cynomolgus monkeys (n= 3),
the plasma concentrations of parent drug 1 and its metabolites were plotted as functions of time,
as shown in Figure 6. Despite the above mentioned concern regarding the metabolic patterns under
in vitro microsomes, a large amount of 1 remained in the plasma (it was the major component),
while small amounts of M1 and M3 and a trace amount of M4 were also observed. However, M2
could not be detected at all after a single oral administration. As a result, we expected that parent
drug 1 would be much more abundant than its metabolites M1, M2, M3 and M4, even in the non-
clinical studies in monkeys.
12

Figure 6. Plasma concentrations of parent drug 1 and its metabolites (M1, M2, M3 and M4) as
functions of time after a single oral administration of 10 mg/kg of 1 to male cynomolgus
monkeys (n=3).
4. Conclusion
We synthesized and identified four metabolites, M1, M2, M3 and M4, generated from parent drug,
1 (K-604) in the liver. From a synthetic perspective, we solved various synthetic problems,
including the regionselective monosulfoxidation of the bis(methylthio) groups and sulfide in the
molecule. To supply sufficient amounts of M1 and M2 in an efficient and regioselective manner,
we catalytically oxidized 1 using a combination of hydrogen peroxide and sodium tungstate by
selecting either phosphoric acid or trifluoroethanol as the solvent for the one-pot synthesis. The
sulfoxidation of 4b with t-BuOCl at -78 °C afforded synthetic intermediate M3 in excellent yield.
The efficient synthesis of M4 was established. Using in vitro experiments, we clarified the profiles
of these metabolites and the metabolic pattern in humans, which was quite similar to that of
13

monkeys. Although a high clearance value of 1 was observed in in vitro metabolic studies, 1 was
much more abundant than M1, M2, M3 and M4 following oral administration of a single dose of
10 mg/kg of 1 to fasted male monkeys.
5. Experimental section
All procedures performed on animals in this study were conducted in accordance with established
guidelines and regulations and were reviewed and approved by the Committee on the Ethics of
Animal Experiments, Kowa Company Ltd.
We computationally checked all metabolites, M1, M2, M3 and M4, for PAINS and found that
they were negative.
5.1. Chemistry
5.1.1. General Methods
Commercially available reagents and solvents were used without further purification. Thin-layer
1
chromatography (TLC) analyses were conducted using silica-gel 60 F254 plates (Merck). H
Nuclear magnetic resonance (NMR) spectra were recorded using a JEOL JNM-LA 400 MHz
instrument. Tetramethylsilane was used as an internal standard for spectra obtained in deuterated
dimethyl sulfoxide (DMSO-d6) and CDCl3. Chemical shifts () are given in parts per million
(ppm), and coupling constants (J values) are given in hertz (Hz) and reported to the nearest 0.1 Hz.
13C-NMR spectra were recorded on a JEOL JNM-LA instrument at 100 MHz in DMSO-d6 and
CDCl3. Infrared (IR) spectra were recorded using a Thermo Nicolet AVATAR370. Mass spectra
were obtained on a JEOL MS-BU20 mass spectrometer (MS). Elemental analyses (C, H, and N)
were performed using a Yanaco MT-5 instrument. Melting points were determined in open glass
capillaries using a Buchi B-545 melting point apparatus. The purities of all the test compounds
14

were greater than 98% based on HPLC chromatograms aquired on an Agilent 1100 series
instrument.
5.1.2. Materials
2-[4-[2-(Benzimidazole-2-ylthio)ethyl]piperazin-1-yl]-N-(2,4-bis(methylthio)-6-methyl-3-
pyridyl)acetamide hydrochloride (1) (K-604), 2-bromo-N-(6-methyl-2,4-bis(methylthio)pyridin-
3-yl)acetamide (5) and 2-(4-(2-hydroxyethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis(methylthio)-
pyridin-3-yl)acetamide (15) were prepared according to our published procedures. 1
2-(4-(2-(Benzo[d]oxazol-2-ylthio)ethyl)piperazin-1-yl)-N-(2-methyl-6-(methylthio)pyridin-3-yl)-
acetamide was used as internal standard (I.S.) for the PK tests. The preparation and the
characterization data of the I. S. are provided in the Supporting Information. The syntheses of the
four major metabolites, M1, M2, M3 and M4, are described below.
5.1.3. Synthetic procedure for compounds M1, M2, M3, and M4.
5.1.4. 2-((2-(Piperazin-1-yl)ethyl)thio)-1H-benzo[d]imidazole Trihydrochloride Salt (4a)
Compound 4a was obtained by deformylation of 4-(2-((1H-benzo[d]imidazol-2-yl)thio)ethyl)-
piperazine-1-carbaldehyde (3a), which was prepared via the coupling of 2-mercaptobenzimidazole
and the methansulfonate of 4-(2-hydroxyethyl)piperazine-1-carbaldehyde (2a) in overall yield of
71% (3 steps) according to the method described in our patent. 17 Colorless crystals. Mp 241−246
1
˚C; IR (KBr) cm-1: 3374, 2938, 2647, 1630, 1522; H-NMR (DMSO-d6) : 3.37−3.57 (8H, m),
3.54 (2H, t, J = 7.0 Hz), 3.81 (2H, t, J = 7.0 Hz), 7.31 (2H, dd, J = 5.9, 3.3 Hz), 7.59 (2H, dd, J =
5.9, 3.3 Hz), 9.73 (2H, br s); Anal. Calcd for C13H21Cl3N4S: C, 42.00; H, 5.69; N, 15.07. Found:
C, 41.87; H, 5.62; N, 14.98.
5.1.5. Alternative synthesis of 3a using a Mitsunobu reaction.
15

The compound 3a was alternatively prepared by the direct coupling between 2-
mercaptobenzimidazole and 2a in 90% yield according to the method described in our patent. 16
Diethyl azodicarboxylate (DEAD, 5.19 g, 29.8 mmol) was added dropwise to a stirred solution of
2a (1.58 g, 10 mmol), 2-mercaptobenzimidazole (5.56 g, 37 mmol) and triphenylphosphine (Ph3P,
9.26 g, 35.3 mmol) in DMF (100 mL) maintained in an ice bath. The reaction mixture was stirred
at the same temperature for 80 min and then quenched by the addition of water (2 mL). The reaction
mixture was concentrated in vacuo. The residue was dissolved in CHCl3 (200 mL). The insoluble
material was removed by filtration through a pad of Celite. The filtrate was concentrated in vacuo.
The residue was partitioned into 2 N HCl (20 mL) and AcOEt (100 mL). The organic layer was
extracted with 2 N HCl (20 mL × 2). The combined aqueous layers were washed with CHCl3 (50
mL × 2) and made alkaline by adding K2CO3, and then extracted with CHCl3 (50 mL × 3). The
organic layer was washed with brine, and dried over Na2SO4. The solvent was evaporated in vacuo.
The residue was crystallized from acetone/ Et2O to yield 3a (2.19 g, 76%) as colorless crystals.
5.1.6. 2-((2-(Piperazin-1-yl)ethyl)thio)-1H-benzo[d]imidazole Tris(trifluoroacetate) (4b)
Compound 4b was obtained by the deprotection of tert-butyl 4-(2-((1H-benzo[d]imidazol-2-
yl)thio)ethyl)piperazine-1-carboxylate (3b), which was prepared via the coupling of 2-
mercaptobenzimidazole and the methansulfonate of tert-butyl 4-(2-hydroxyethyl)piperazine-1-
carboxylate (2b) in an overall yield of 42% (3 steps) according to the method described in our
patent. 17 The product was obtained as pale-yellow crystals. Mp 146−148 ˚C; IR (KBr) cm-1: 3572,
3512, 1675, 1619, 1536, 1189, 1132; 1H-NMR (DMSO-d6) : 3.05 − 3.11 (4H, m), 3.15 (2H, t, J
= 6.3 Hz), 3.19 – 3.27 (4H, m), 3.55 (2H, t, J = 6.3 Hz), 7.16 −7.22 (2H, m), 7.46 – 7.52 (2H, m),
8.75 – 9.10 (2H, m); Anal. Calcd for C19H21F9N4O6S: C, 37.75; H, 3.50; N, 9.27. Found: C, 37.56;
H, 3.67; N, 9.20.
16

5.1.7. 2-Bromo-N-(6-methyl-4-(methylsulfinyl)-2-(methylthio)pyridin-3-yl)acetamide (6)
Sodium perborate (NaBO3, 6.22 g, 40.4 mmol) was added portion-wise to a stirred suspension of
5 (10.0 g, 31.1 mmol) in AcOH (600 mL) in an ice bath. After stirring for 30 min, the reaction
mixture was allowed to warm to rt, it was stirred for an additional 15 h, and then it was concentrated
in vacuo. The residue was purified by silica gel column chromatography (SiO2; 200 g) and eluted
with CHCl3/sat. NH3−MeOH (50:1 to 10:1) to afford a pale-yellow solid, which was further
purified by silica gel column chromatography (SiO2; 100 g) and eluted with CHCl3/sat.
NH3−MeOH (10:1) to yield a colorless solid, which was washed with Et2O to give the title
compound (6) as a colorless amorphous solid (676 mg, 6%). IR (KBr) cm-1: 3467, 3145, 2969,
1693, 1560, 1522, 1416, 1041; 1H−NMR (DMSO−d6) : 2.49 (3H, s), 2.58 (3H, s), 2.71 (3H, s),
4.05 (2H, s), 7.39 (1H, s), 10.2 (1H, br s).
5.1.8. 2-(4-(2-((1H-Benzo[d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-4-
(methylsulfinyl)-2-(methylthio)pyridin-3-yl)acetamide (M1)
K2CO3 (480 mg, 3.37 mmol) and a solution of 6 (261 mg, 0.77 mmol) in DMF (9 mL) were
sequentially added to a stirred solution of 4b (465 mg, 0.77 mmol) in DMF (18 mL) at rt. The
reaction mixture was stirred at rt for 1.5 h, and then diluted with water (50 mL). The organic layer
was separated. The aqueous layer was extracted with AcOEt (100 mL × 3). The combined organic
layers were washed with water (150 mL × 3) and brine (150 mL), and dried over Na2SO4. The
solvent was evaporated in vacuo. The residue was purified by preparative TLC developed with
CHCl3/sat. NH3−MeOH (20:1) to yield a colorless solid (322 mg, 80%), which was recrystallized
from n-hexane/AcOEt to give the title compound (M1, 234 mg, 58%) as colorless crystals. Mp.
121−124 ˚C; IR (KBr) cm-1: 3429, 3182, 2924, 2817, 1670, 1479, 1440, 1403; 1H−NMR (DMSO-
d6) : 2.47 (3H, s), 2.50 (3H, s), 2.53−2.62 (9H, m), 2.68−2.73 (1H, m), 2.71 (3H, s), 3.08 (1H, d,
17

J = 15.6 Hz), 3.14 (1H, d, J = 15.6 Hz), 3.42 (2H, t, J = 6.8 Hz), 7.06−7.15 (2H, m), 7.32−7.51
(3H, m), 9.51 (1H, s), 12.55 (1H, br s); 13C-NMR (100 MHz, DMSO-d6) δ: 12.38, 24.17, 28.73,
41.91, 52.17 (2C), 52.92 (2C), 57.17, 61.03, 112.23, 117.20, 121.25 (2C), 123.60 (2C), 143.74,
154.27 (2C), 157.57, 158.03, 169.76; Anal. Calcd for C23H30N6O2S3: C, 53.26; H, 5.83; N, 16.20;
S, 18.54. Found: C, 53.02; H, 5.79; N, 16.12; S, 18.40. HPLC purity: 98.31 ％ . column:
CAPCELPAK C18 UG120; column size 5 m, 4.6 mm × 150 mm; mobile phase 10 mM SLS/5
mM H3PO4 in MeCN = 50:50; flow rate 1.0 mL/min; column temperature 40 °C; wavelength 247
nm; retention time 5.67 min.
5.1.9. Alternative synthesis for the large scale preparation of M1.
To a stirred solution of methytri(n-octyl)ammonium]hydrogen sulfate (Me(n-Oct)3NHSO4, 9.4
mg, 0.02 mmol) in 30% hydrogen peroxide (H2O2, 2.5 g, 22 mmol) was added sodium tungstate
dihydrate (Na2WO4·2H2O, 6.6 mg, 0.02 mmol) at 0 ˚C. The resulting mixture was stirred at the
same temperature to afford a homogeneous solution. Compound 1 (10 g, 18.5 mmol) was dissolved
in a mixture of 90% phosphoric acid (100 mL) and conc. H2SO4 (15 mL) with heating. To this
solution was added the prepared H2O2 solution at 0 ˚C over 2 min. After stirring for 20 h at rt, the
reaction mixture was carefully poured into a mixture of ice and excess NaHCO3. The mixture was
neutralized with NaHCO3 and extracted with CH2Cl2 (200 mL × 3). The organic layer was washed
with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel
column chromatography (SiO2; 1000 g, CHCl3/sat. NH3−MeOH = 50:1) to yield a solid, which
was crystallized from CHCl3 and Et2O to give the title compound (M1, 4.49 g, 47%) as colorless
crystals.
18

5.1.10. 2-(4-(2-((1H-Benzo[d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2-(methyl-
sulfinyl)-4-(methylthio)pyridin-3-yl)acetamide (M2)
Step 1. A solution of m-chloroperbenzoic acid (m−CPBA, 65% purity, 501 mg, 1.88 mmol) in
CHCl3 (40 mL) was added dropwise to a stirred suspension of 5 (500 mg, 1.56 mmol) in CHCl3
(40 mL) in an ice bath over 5 min. After stirring for 15 min at the same temperature, the reaction
mixture was concentrated in vacuo to give a crude mixture of 7 as a solid. Compound 7 was not
stable enough to isolate and characterize, and thus the crude material was dissolved in MeCN (30
mL) for use in the next step without purification.
Step 2. The thus-prepared solution of 7 in MeCN (30 mL) was added to a stirred solution of 4b
(943 mg, 1.56 mmol) and K2CO3 (1.38 g, 9.98 mmol) in MeCN (50 mL) in an ice bath. The reaction
mixture was stirred at rt for 14 h and then filtered through a pad of Celite. The filtrate was
concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted
with CHCl3/sat. NH3-MeOH (10:1 to 4:1) to yield a yellow solid (268 mg, 33%), which was
purified from n-hexane/AcOEt to give the title compound (M2, 168 mg, 21%) as a colorless
amorphous solid. IR (KBr) cm-1: 3146, 2935, 2814, 1733, 1694, 1571, 1480, 1437, 1036; 1H-NMR
(DMSO-d6) : 2.48−2.52 (6H, m), 2.53−2.62 (9H, m), 2.68−2.74 (4H, m), 3.08 (1H, d, J = 15.9
Hz), 3.19 (1H, d, J = 15.9 Hz), 3.43 (2H, t, J = 7.2 Hz), 7.07−7.14 (2H, m), 7.31−7.52 (3H, m),
13
9.59 (1H, s), 12.58 (1H, br s); C-NMR (100 MHz, DMSO-d6) δ: 13.36, 23.83, 28.76, 38.54,
52.25 (2C), 52.92 (2C), 57.20, 60.90, 117.23, 120.51, 121.31 (2C), 125.45 (2C), 143.81, 150.47
(2C), 150.61, 157.67, 157.82, 169.94; Anal. Calcd for C23H30N6O2S3·0.2AcOEt·0.1H2O: C, 53.12;
H, 5.96; N, 15.62, S, 17.88. Found: C, 53.03; H, 5.91; N, 15.33; S, 17.89. HPLC purity: 98.68%
column: CAPCELPAK C18 UG120; column size 5 m, 4.6 mm × 150 mm; mobile phase 10 mM
19

SLS/5 mM H3PO4 in MeCN= 45:55; flow rate 1.0 mL/min; column temperature 40 °C; wavelength
247 nm; retention time 6.78 min.
5.1.11. Alternative synthesis for the large scale preparation of M2
To a solution of Me(n-Oct)3NHSO4 (47 mg, 0.1 mmol) in 30% H2O2 (7.5 g, 66 mmol) was added
Na2WO4 (33 mg, 0.1 mmol) at 0˚C. The resulting mixture was stirred at same temperature to
prepare a homogeneous solution. This solution (2.1 mL) was added to a stirred solution of 1 (50 g,
92.5 mmol) in MeOH (500 mL) at 0˚C. After stirring for 24 h at rt, solutions of sat. aq. Na2SO3
(50 mL) and sat. aq. K2CO3 (50 mL) were sequntially added to the reaction mixture. The mixture
was extracted with CH2Cl2 (1000 mL). The organic layer was washed with brine, dried over
Na2SO4, and then concentrated in vacuo. The residue was purified by silica gel column
chromatography (SiO2; 1000 g, CHCl3/sat. NH3−MeOH = 100:1 to 50:1) to yield a solid, which
was further purified to give the compound (M2, 24.75 g, 52%) as a colorless amorphous solid.
5.1.12. 2-(4-(2-((1H-Benzo[d]imidazol-2-yl)sulfinyl)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-
bis(methylthio)pyridin-3-yl)acetamide (M3)
Tert-butyl hypochlorite (t-BuOCl, 0.374 mL, 3.31 mmol) was dropwise added to a stirred solution
of 4b (2.00 g, 3.31 mmol) in MeOH (20 mL) over 2 min at -78 °C. The reaction mixture was
stirred at the same temperature for 1 h. A saturated solution of Na2CO3 (2 mL) was added to this
mixture. The reaction mixture was allowed to warm to rt and stirred for an additional 13 h. The
solvent was evaporated in vacuo. The residue was purified by silica gel column chromatography
(SiO2; 100 g, CHCl3/sat. NH3−MeOH = 5:1) to yield the title compound (10, 920 mg, 99%) as a
1
colorless solid. IR (KBr) cm-1: 3410, 2951, 2820, 1629, 1447, 1371, 1032; H-NMR (CDCl3) :
2.34−2.60 (4H, m), 2.68−2.82 (4H, m), 2.87 (1H, ddd, J = 13.6, 7.4, 5.5 Hz), 3.00 (1H, ddd, J =
20

13.6, 6.6, 5.6 Hz), 3.35 (1H, ddd, J = 13.8, 6.6, 5.5 Hz), 3.49 (1H, ddd, J = 13.8, 7.4, 5.6 Hz),
7.30−7.37 (2H, m), 7.63−7.72 (2H, m); EIMS m / z 278 (M+).
A solution of 5 (321 mg, 1.0 mmol) in MeCN (50 mL) was added to a stirred solution of K2CO3
(207 mg, 1.50 mmol) and 10 (278 mg, 1.0 mmol) in MeCN (50 mL) at rt. The reaction mixture
was stirred at rt for 6 h, and then filtered through a pad of Celite. The filtrate was concentrated in
vacuo. The residue was purified by silica gel column chromatography (SiO2; 40 g, CHCl3/sat.
NH3−MeOH = 25:1) to yield a solid (450 mg, 87%), which was recrystallized from n-
hexane/Et2O/acetone to give the title compound (M3, 359 mg, 69%) as colorless crystals. Mp
121−126 ˚C; IR (KBr) cm-1: 3447, 3143, 2923, 2814, 1685, 1571, 1563, 1530; 1H-NMR (DMSO-
d6) : 2.37 (3H, s), 2.38 (3H, s), 2.43 (3H, s), 2.71 (1H, ddd, J = 13.2, 7.3, 6.4 Hz), 2.76 (1H, ddd,
J = 13.2, 7.3, 6.4 Hz), 2.93 (2H, s), 3.29–3.51 (10H, m), 6.87 (1H, s), 7.26–7.31 (2H, m), 7.59–
13
7.66 (2H, m), 9.07 (1H, s), 12.46 (1h, br s); C-NMR (100 MHz, DMSO-d6) δ: 12.19, 13.11,
24.03, 49.10, 50.94, 52.06 (2C), 52.64 (2C), 61.07, 113.81, 120.51, 122.95 (2C), 124.19 (2C),
149.09, 154.68 (2C), 155.82, 156.04, 168.48. Anal. Calcd for C23H30N6O2S3: C, 52.36; H, 5.83;
N, 16.20, S, 18.54. Found: C, 53.02; H, 5.76; N, 16.20; S, 18.63. HPLC purity: 98.99%; column:
CAPCELPAK C18 UG120; column size 5 m, 4.6 mm × 150 mm; mobile phase 10 mM SLS/5
mM H3PO4 in MeCN= 50:50; flow rate 1.0 mL/min; column temperature 40 °C; wavelength 247
nm; retention time 4.91 min.
5.1.13. 2-(4-(2-((5-Hydroxy-1H-benzo[d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-
2,4-bis(methylthio)pyridin-3-yl)acetamide (M4)
Step 1. K2CO3 (26.9 g, 195 mmol) and benzyl bromide (28.4 g, 166 mmol) were sequentially added
to a stirred solution of 4-amino-3-nitrophenol (11, 25.0 g, 162 mmol) in acetone (350 mL) in an
21

ice bath. The reaction mixture was allowed to warm to rt and was stirred for another 18 h. The
solvent was evaporated in vacuo. The residue was dissolved in water (500 mL) and AcOEt (700
mL). The organic layer was separated. The aqueous layer was extracted with AcOEt (300 mL ×
2). The combined organic layers were washed with water (500 mL), brine (300 mL) and dried over
Na2SO4. The solvent was evaporated in vacuo. The residue was purified by silica gel column
chromatography (SiO2; 1000 g, n-hexane/acetone = 7:1 → 5:1 → 3:1 → 2:1) to yield 4-benzyloxy-
2-nitroaniline (12, 34.2 g, 86%) as red crystals. IR (KBr) cm-1: 3476, 3351, 1642, 1593, 1511,
1415, 1250, 1206, 1154, 1015; 1H-NMR (CDCl3) : 5.03 (2H, s), 5.88 (2H, br s), 6.76 (1H, d, J =
9.1 Hz), 7.13 (1H, dd, J = 9.1, 2.9 Hz), 7.31-7.45 (5H, m), 7.65 (1H, d, J = 2.9 Hz).
Step 2. Zinc dust (18.8 g, 288 mmol) was added portionwise to a solution of 12 (20.0 g, 81.9 mmol)
and conc. HCl (20 mL) in AcOH (600 mL) in an ice bath. The reaction mixture was allowed to
warm to rt, and was then stirred for 1 h. The solvent was evaporated in vacuo. The residue was
diluted with water (500 mL) and 5 M NaOH (200 mL) was added to make it alkaline (pH = 10).
The insoluble material was removed by filtration through a pad of Celite. The filtrate was extracted
with CHCl3 (1500 mL × 2, 1000 mL). The combined organic layers were washed with water (800
mL) and brine (500 mL), and dried over Na2SO4. The solvent was evaporated to yield crude 4-
(benzyloxy)benzene-1,2-diamine (13, 16.2 g, 92%) as a dark red oil.
Step 3. Potassium ethylxanthogenate (19.7 g, 123 mmol) was added to a stirred solution of
compound 13 (16.2 g, 75.6 mmol) in EtOH (1000 mL) at rt. The reaction mixture was stirred at
reflux for 7 h. The solvent was evaporated in vacuo. The residue was diluted with water (1000 mL)
and conc. HCl (15 mL) was added to make it acidic (pH = 3). The insoluble material was removed
by filtration through a pad of Celite. The filtrate was extracted with AcOEt (500 mL × 2). The
combined organic layers were washed with water (800 mL) and brine (500 mL), and dried over
22

Na2SO4. The solvent was evaporated in vacuo. The residue was purified by silica gel column
chromatography (SiO2; 1000 g, CHCl3/sat. NH3−MeOH = 100:3) to yield 5-(benzyloxy)-1H-
benzo[d]imidazole-2-thiol (14, 12.9 g, 62%), which was recrystallized from n-hexane/acetone to
give pale-yellow crystals (9.95 g, 51%). IR (KBr) cm-1: 3132, 3092, 1635, 1593, 1498, 1452, 1270,
1
1114, 818; H-NMR (DMSO-d6) : 3.37 (1H, br s), 5.09 (2H, s), 6.75 (1H, d, J = 2.4 Hz), 6.80
(1H, dd, J = 8.5, 2.4 Hz), 7.03 (1H, d, J = 8.5 Hz), 7.28 – 7.46 (5H, m), 12.38 (1H, d, J =11.7 Hz).
Step 4. Methansulfonyl chloride (MsCl, 3.99 g, 34.7 mmol) was added dropwise to a stirred
solution of 2-(4-(2-hydroxyethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis-(methylthio)pyridin-3-
yl)acetamide (15, 9.19 g, 24.8 mmol), Et3N (3.02 g, 29.8 mmol) and DMAP (303 mg, 2.48 mmol)
in THF (200 mL) in an ice bath. The reaction mixture was stirred at the same temperature for 30
min. The insoluble material was removed by filtration. The filtrate was concentrated in vacuo. The
residue was dissolved in DMF (120 mL). This solution was added slowly to a stirred solution of
K2CO3 (5.14 g, 37.2 mmol), 18-crown-6 (656 mg, 2.48 mmol) and 14 (7.00 g, 27.3 mmol) in DMF
(200 mL) at rt. The reaction mixture was stirred at 80 °C for 5 h and then diluted with water (1000
mL) and AcOEt (400 mL). The organic layer was separated. The aqueous layer was extracted with
AcOEt (400 mL × 2). The combined organic layers were washed with water (1000 mL × 3) and
brine (500 mL) and dried over Na2SO4. The solvent was evaporated in vacuo. The residue was
purified by silica gel column chromatography (SiO2; 1000 g, n-hexane/acetone = 30:1 to
CHCl3/MeOH
=
20:1)
to
yield
2-(4-(2-((5-(benzyloxy)-1H-benzo[d]imidazol-2-
yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis(methylthio)pyridin-3-yl)acetamide (16, 13.3 g,
88%) as a pale-yellow solid. IR (KBr) cm-1: 3243, 2924, 2361, 1682, 1484, 1339, 1157, 1011, 806;
1H-NMR (CDCl3) : 2.42 (3H, s), 2.49 (3H, s), 2.52 (3H, s), 2.76 – 3.02 (10H, m), 3.18 – 3.24
23

(2H, m), 3.31 (2H, s), 5.10 (2H, s), 6.66 (1H, s), 6.92 (1H, dd, J = 8.8, 2.5 Hz), 7.23 – 7.48 (6H,
m), 8.41 (1H, br s).
5.1.14. Alternative synthesis of 16 using a Mitsunobu reaction
Compound 16 was alternatively prepared by the direct coupling between 14 and 15 in a yield of
95% according to the method described in our patent. 32
Step 5. Thioanisole (63.1 g, 508 mmol) was added to a stirred solution of 16 (7.73 g, 12.7 mmol)
in CF3COOH (80 mL). The reaction mixture was stirred at 50 °C for 3 h. After completion of the
reaction, the solvent was evaporated in vacuo. The residue was purified by silica gel column
chromatography (SiO2; 100 g, CHCl3/MeOH = 10:1 to CHCl3/sat. NH3−MeOH = 20:1 ) to yield
the title compound (M4, 6.45 g, 98%) as a pale-yellow solid, which was recrystallized from n-
hexane/Et2O/CHCl3 to give colorless crystals (4.54 g, 68%). Mp 142 – 144 °C; IR (KBr) cm-1:
3297, 2923, 2825, 1707, 1627, 1562, 1481; 1H-NMR (CDCl3) : 2.42 (3H, s), 2.51 (3H, s), 2.53
(3H, s), 2.66 – 3.02 (10H, m), 3.18 (2H, t, J = 5.1 Hz), 3.32 (2H, s), 6.69 (1H, s), 6.77 (1H, dd, J
= 8.5, 2.5 Hz), 6.96 (1H, br s), 7.36 (1H, br s); 13C-NMR (100 MHz, DMSO-d6) δ: 12.21, 13.13,
24.05, 28.94, 52.26 (2C), 52.93 (2C), 53.71, 61.19, 102.53, 110.34, 113.83, 117.55, 117.75,
136.21, 137.43, 147.64, 149.12, 155.84, 156.07, 168.57; Anal. Calcd for C23H30N6O2S3·0.2H2O
C, 52.89; H, 5.87; N, 16.09, S, 18.42. Found: C, 52.86; H, 5.76; N, 15.99; S, 18.25. HPLC purity:
99.19%. column: CAPCELPAK C18 UG120; column size 5 m, 4.6 mm × 150 mm; mobile phase
10 mM SLS/5 mM H3PO4 in MeCN = 52:48 (0 - 15 min), 30:70 (15 - 40 min); flow rate 1.0
mL/min; column temperature 40 °C; wavelength 247 nm; retention time 6.06 min.
6. Biology
6.1. Determination of four metabolites M1, M2, M3, and M4, usng human liver microsomes
24

Parent compound 1 (K-604) was incubated with human liver microsomes (0.05 mg protein/mL)
for 5 min at 37 ℃ in the presence of NADPH (0.25 mg of β-NADP, 0.25 mg of G-6-P, and 0.1U
of G6PDH). After extraction with a solid phase extraction column (Waters Oasis HLB 1 cc (30
mg)), the sample was analyzed by LC/MS/MS. The LC/MS/MS system was equipped with an
HPLC: Waters 2690, MS: TSQ-7000 (Thermo Quest) system with a reversed-phase column
(Inertsil ODS-3, 3.0 × 150 mm, Ф 5 μm; GL Science Inc.). The column temperature was 40 ℃,
the mobile phase was 0.1% NH4HCOO (pH 4)/MeCN/MeOH (55:35:10, v/v/v), and the flow rate
was 0.3 mL / min. The parent mass ions (m/z) of 1 (K-604), M1, M2, M3, M4 and the I. S. were
502.9, 518.9, 518.9, 518.9, 518.9 and 471.9, respectively and fragment ions were 352.7, 368.7,
368.7, 352.9, 352.9, and 320.9, respectively.
6.2. Assessment of the metabolic pattern using human and other species’ microsomes by HPLC-
radioluminography
HPLC-radioluminography was employed for determination using 14C-K-604. Specifically, the
sample was separated by HPLC, and the eluate was collected in a 96-well microplate with 15
seconds per well. After drying overnight, the microplate was contacted with the imaging plate for
approximately 12 h. The result was analyzed by a bioimaging analyzer (BAS-2500, Fuji Photo
Film).
6.3. Monkey study design
In vivo drug absorption and PK studies were performed as follows. Cynomolgus (purpose-bred)
male monkeys (source: Indonesia; 6 years old; body weight 5 kg to 6 kg) were used for the in vivo
studies. The monkeys were obtained from LSI Medience Corporation (Kashima, Ibaraki, Japan).
All procedures performed on animals in this study were in accordance with established guidelines
25

and regulations, and were reviewed and approved by the Committee on the Ethics of Animal
Experiments, Kowa Company Ltd. The monkeys were fed a commercially prepared monkey diet
(PS type, Oriental Yeast Co., Ltd., Chiba, Japan) and housed in cages under uniform and
environmentally controlled conditions.
6.4. Measurement of the Plasma Contents of Parent Drug 1 and its Metabolites, M1, M2, M3 and
M4, after a Single Oral Administration of 1 to Male Cynomolgus Monkeys
A solution (1 mL/kg) of 1 (10 mg K-604/1 mL in 0.06 N HCl) was administered into the stomach
using a nasogastric catheter to three male monkeys (body weight 5 kg to 6 kg). Using a syringe
treated with heparin sodium, plasma samples (1 mL of blood/collection) were collected from the
femoral veins of male cynomolgus monkeys at 15, 30, 60, 120, 180, 240, and 360 min after
administration. The blood was centrifuged at 3,000 rpm (1710 g) for 15 min at 4 °C to obtain the
plasma. Plasma samples were immediately frozen in a deep freezer (acceptable range: -70 °C or
below). The PK plasma samples were treated according to a validated method for analyzing
monkey’s plasma. The IS-100 was prepared by adding methyl tert-butyl ether (MTBE) (1 mL) to
blank samples. The IS-100 solution (1 mL) and MTBE (5 mL) were added to the PK samples. The
resulting mixture was mechanically shaken for 10 min at rt and centrifuged at 2500 rpm for 5 min
at 20 °C. The organic layer was transferred into a separate test tube and concentrated under a
stream of nitrogen at 40 °C in a water bath. The residue was dissolved in mobile phase-1 (0.25
mL). The sample solution was stirred well and transferred into a glass HPLC vial. The sample
solution (0.1 mL) was injected into HPLC system. Stability tests confirmed that the extracted
plasma samples were stable for 96 h if they were kept at 4 °C. The concentrations of K-604 and
its metabolites in the plasma samples were determined by the column-switching HPLC method
26

(CLASS-LC 10, Shimadzu Corporation, Kyoto, Japan). The column-switching HPLC analysis was
performed with Inertsil ODS-3 Guard column (10 × 4 mm; GL Sciences Inc., Tokyo, Japan) for
preseparation and a YMC Hydrosphere C18 column (150 × 4.6 mm; YMC Co., Ltd., Kyoto, Japan)
for analytical separation. Two mobile phases, 10 mmol/L phosphate buffer (pH 2.8) and MeCN
(63:37 (v/v), mobile phase-1) for the pre-separation and 10 mmol/L phosphate buffer (pH 2.8) and
MeCN (58:42 (v/v), mobile phase-2) were used for the analytical separation, and the flow-rate was
maintained at 1 mL/min. Mobile phase-1 was delivered to the preseparation column using Pump
A and mobile phase-2 was delivered to the preseparation column using Pump B. The UV detection
wavelength was 289 nm, the column temperature was maintained at 40 ℃. The run time was 30
min, the time for switching the column was set at 7.5 min. The HPLC system (L-6000; Hitachi,
series) was equipped with a UV detector (254 nm), a reversed-phase column (Cosmosil 5C18, 4.6
mm × 150 mm, Ф5 μm; Nacala Tesque) and automatic injector (AS-4000; Hitachi)) was used. The
retention times for 1 (K-604), M1, M2, M3, M4 and the I.S. were 11.0, 15.5, 12.1, 14.6, 17.2 and
25.7 min, respectively.
ASSOCIATED CONTENT
A. Supplementary data
Selected reaction monitoring (SRM) chromatogram of human liver microsomes spiked with
standards (K-604, M1, M2, M3, and M4.; HPLC-RLG radio chromatogram of the metabolic
reaction of K-604;in hepatic human and rats microsomes.; detailed experimental procedures for
the syntheses of [piperazine-2,3(5,6)-14C] K-604 and I. S..
27

AUTHOR INFORMATION
Corresponding Author
*E-mail: t-ogiya@kowa.co.jp Phone: (+81) 42-391-6211. Fax: (+81) 42-395-0312.
ORCID
Tadaaki Ohgiya: 0000-0002-3068-3610.
Author Contributions
All authors have given approval to the final version of the manuscript and declare that they have
no conflict of interest.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We appreciate Dr. Ayumu Okuda for calculating the electron densities on the bis(methylthio)
groups in the model structures 8 and 9 using 6-31G**. We also acknowledge Drs. Gen Watanabe
and Takaaki Araki for proofreading the manuscript.
ABBREVIATIONS
ACAT, acyl-CoA:cholesterol O-acyltransferase; AUC, area under the curve; CL, clearance;
Cmax, maximum drug concentration; CYP, cytochrome P; Km, Michaelis constant; Tmax,
maximum drug concentration time; Vmax, maximal velocity.
References
1. Shibuya, K.; Kawamine, K.; T.; Ozaki, C.; Ohgiya, T.; Edano, T. Yoshinaka, Y.; Tsunenari, T.
Discovery of clinical candidate 2-(4-(2-((1H-benzo[d]imidazol-2-yl)- thio)ethyl)piperazin-1-
28

yl)-N-(6-methyl-2,4-bis(methylthio)pyridin-3-yl)acetamide hydrochloride [K-604], an aqueous
soluble acyl-CoA:cholesterol O-acyltransferase (ACAT/SOAT)-1 inhibitor. J. Med. Chem.
2018, 61, 10635-10650.
2. Shibuya, K.; Kawamine, K.; Sato, Y.; Miura, T.; Ozaki, C.; Edano, T.; Hirata, M. Novel cyclic
diamine compounds and medicine containing the same. WO1998054153, May 26, 1998.
3. Shibuya, Y.; Chang, C. C. Y.; Huang, L-H.; Bryleva, E. Y.; Chang, T-Y. Inhibiting ACAT1/
SOAT1 in microglia stimulates autophagy-mediated lysosomal proteolysis and increases A1-
42 clearance. J. Neurosci. 2014, 34, 14484-14501.
4. Yamanaka, K.; Urano, Y.; Takabe, W.; Saito, Y.; Noguchi, N. Induction of apoptosis and
necroptosis by 24(S)-hydroxycholesterol is dependent on activity of acyl-CoA:cholesterol
acyltransferase 1. Cell Death Dis. 2014, 5, e990.
5. Shibuya, Y.; Chang, C. C. Y.; Chang, T-Y. ACAT1/SOAT1 as a therapeutic target for
Alzheimer’s disease. Future Med. Chem. 2015, 7, 2451-2467.
6. Shibuya, K.; Watanabe, T.; Urano, Y.; Takabe, W.; Noguchi, N.; Kitagishi, H. Synthesis of
24(S)-hydroxycholesterol esters responsible for the induction of neuronal cell death. Bioorg.
Med. Chem. 2016, 24, 2559-2566.
7. Takabe, W.; Urano, Y.; Vo, D-K. H.; Shibuya, K.; Tanno, M.; Kitagishi, H.; Fujimoto, T.;
Noguchi, N. Esterification of 24S-OHC induces formation of atypical lipid droplet-like
structures, leading to neuronal cell death. J. Lipid. Res. 2016, 57, 2005-2014.
8. Shibata, H.; Shibuya, K. An ACAT-1 inhibitor, K-604, ameliorates hepatic inflammation in
NAFLD and NASH models. Gastroenterol. Pancreatol. Liver Disord. 2016, 3, 1-11.
9. Kola, I.; Landis, J. Can the pharmaceutical industry reduce attrition rates? Nat. Rev. Drugs
Discov. 2004, 3, 711-715.
29