Discovery of Clinical Candidate 2-(4-(2-((1 H-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 Aqueous-Soluble Acyl-CoA:Cholesterol O-Acyltransferase-1 Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.8b01256

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, 2) has been identified as an aqueous-soluble potent inhibitor of human acyl-coenzyme A:cholesterol O-acyltransferase

Full text of DOI:10.1021/acs.jmedchem.8b01256

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Article
Discovery of the 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 hydrocholoride [K-604], an Aqueous Soluble Acyl-
CoA: Cholesterol O-Acyltransferase (ACAT/SOAT)-1 Inhibitor
Kimiyuki Shibuya, Katsumi Kawamine, Chiyoka Ozaki, Tadaaki Ohgiya,
Toshiyuki Edano, Yasunobu Yoshinaka, and Yoshihiko Tsunenari
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01256 • Publication Date (Web): 15 Nov 2018
Downloaded from http://pubs.acs.org on November 16, 2018
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Discovery of the 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 [K-604], an Aqueous-Soluble Acyl-
CoA:Cholesterol O-Acyltransferase (ACAT/SOAT)-
1 Inhibitor
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Kimiyuki Shibuya*, Katsumi Kawamine, Chiyoka Ozaki, Tadaaki Ohgiya, Toshiyuki Edano,
Yasunobu Yoshinaka, Yoshihiko Tsunenari.
Tokyo New Drug Research Laboratories, Pharmaceutical Division, Kowa Company, Ltd., 2-17-
43, Noguchicho, Higashimurayama, Tokyo 189-0022, Japan
KEYWORDS. acyl-CoA:cholesterol O-acyltransferase (ACAT/SOAT), ACAT-1 (aortic
ACAT), ACAT-2 (intestinal ACAT), isoform selectivity, cholesteryl esters (CEs), lipid
accumulation areas, atherosclerosis, aqueous soluble, well absorbed, adrenal toxicity, well
tolerated.
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ABSTRACT. K-604 (2) (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) has been identified as an
aqueous-soluble potent inhibitor of human acyl-coenzyme A:cholesterol O-acyltransferase
(ACAT, also known as SOAT)-1 and exhibits 229-fold selectivity for human ACAT-1 over
human ACAT-2. In our molecular design, the insertion of a piperazine unit in the place of a 6-
methylene chain into the linker between the head (pyridylacetamide) and tail (benzimidazole)
moieties led to a marked enhancement of the aqueous solubility (up to 19 mg/mL at pH 1.2) and
a significant improvement of the oral absorption (the Cmax of 2 was 1100-fold higher than that
of 1 in fasted dogs) compared to the previously selected compound 1. After ensuring the
pharmacological effects and safety, we designated 2 as a clinical candidate, named K-604.
Considering the therapeutic results of ACAT inhibitor in past clinical trials, we believe that K-
604 will be useful for the treatment of incurable diseases involving ACAT-1 overexpression.
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Introduction
Acyl-coenzyme A:cholesterol O-acyltransferase (ACAT), also known as sterol O-
acyltransferase (SOAT), has received attention as a promising target for hyperlipidemia and
atherosclerosis. ACAT catalyzes the acylation of cholesterol with long-chain fatty acids to afford
cholesteryl esters (CEs) and is involved in intestinal absorption of dietary cholesterol,
incorporation of hepatic cholesterol into very low-density lipoproteins (VLDLs) and vascular
1
accumulation of cholesterol.
Therefore, exciting and intensive research activity in both
academia and the industry over the last three decades has produced a large number of ACAT
inhibitors as poststatin (a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor) targets; 2-5
however, the beneficial effects of these inhibitors have not been demonstrated in clinical trials. 6-
10
Two isoforms of ACAT have been identified in mammals and have been shown to have
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different substrate specificities and potential functions. ACAT-1 is ubiquitously expressed in
various human tissues, predominantly in human liver (Kupffer cells), macrophage, adrenal gland
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11-17
and brain tissue, whereas ACAT-2 is found in human hepatocytes and intestinal tissue.
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Recently, an ACAT-1 inhibitor was highlighted in the context of drug repositioning
(repurposing) for potential application in the treatment of the following diseases: 1) In multiple
neurodegenerative diseases, including Alzheimer’s disease, 18-22 blocking ACAT-1 provides new
benefits, such as clearance of amyloid  (A) peptides and suppression of 24(S)-
23
hydroxycholesterol-induced neuronal cell death. 2) In nonalcoholic steatohepatitis (NASH),
progression of steatosis and liver inflammation via Kupffer cells activated by high levels of CE
is involved in ACAT-1 induction. Therefore, ACAT-1 inhibition may be a new therapeutic target
for the treatment of NASH. 3) Increased CE levels are widely known to be associated with breast
24-27
cancer, leukemia, glioma and prostate cancer.
P. Saraon and his group have proposed that
ACAT-1 expression could serve as a potential prognostic marker for prostate cancer, specifically
to differentiate between indolent and aggressive forms of cancer. J. Li et al. demonstrated that
ACAT-1 inhibition-induced ER stress led to apoptosis in pancreatic cancer cells. C. Xu and
coworkers revealed that ACAT-1 inhibition potentiated the antitumor response of CD8+ T cells
by modulating cholesterol metabolism. The researchers also demonstrated that ACAT-1
inhibitors can complement current therapies such as immune checkpoint blockade by acting via a
different mechanism. These fascinating trends prompted us to report our discovery of the 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 (2) (K-604), 28-31 an aqueous soluble ACAT-1
inhibitor developed from our lead compound 9-(benzo[d]oxazol-2-ylthio)-N-(2,6-
diisopropylphenyl)nonanamide (1), 32 as shown in Figure 1.
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S
S
H
N
N
N
N
O
N
H
N
O
S
S
N
N
H
HCl
O
9
2
1
(K-604)
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Rabbit aortic ACAT
Rabbit intestinal ACAT IC50 = 0.45 M
Human ACAT-1
Human ACAT-2
LogP 3.3 at pH 6.8
IC50 = 0.084 M
Rabbit aortic ACAT
Rabbit intestinal ACAT IC50 = 0.021
cLogP 7.25
IC50 = 0.004
M
M
IC50 = 0.45 M
IC50 = 102.85 M
Aqueous solubility 0.03
g/mL at pH 1.2
Aqueous solubility 19 mg/mL at pH 1.2
Figure 1. Biological and physical properties of 2 (K-604), developed from our lead compound 1
32
In a previous paper,
we reported our molecular design based on a double induced fit
33
mechanism
for the feasible development of the aortic-selective ACAT inhibitor 1 and
examined the pharmacological profile of this compound. Although 1 was efficacious when orally
administered to high-fat- and cholesterol-fed F1B hamsters, there remained room to considerably
decrease the lipophilicity of this compound to increase aqueous solubility and consequently
improve oral absorption and acceptable safety for further development of a clinical candidate. To
34, 35
address these issues, we used a solubility-driven structural optimization strategy;
specifically, we focused on significant improvement of the physical properties via a small
structural modification so that we could follow our established molecular design composed of
two structurally different ligands (arylacetamide and benzazole) connected with a linker. We first
focused on the structural accommodation of a linker moiety and the incorporation of basic
amines into the molecule. In this paper, we report the molecular design strategy used to obtain an
aqueous-soluble ACAT-1 inhibitor, 2 (K-604), as a clinical candidate, and we describe the
structure-activity relationships (SARs), pharmacological properties, pharmacokinetic (PK)
properties and safety test results of this compound.
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Chemistry
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To investigate the desired linker structure, the spatial arrangement and optimal bridging
distance between the N-(2,6-diisopropylphenyl)acetamide unit (A) and the 2-
(methylthio)benzo[d]oxazole unit (B) for maintenance of potency, and the intestinal IC50/aortic
IC50 selectivity ratio, we prepared the biological compounds for investigation as follows.
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H
O
N
N
O
N
H
N
S
S
Linker
+
O
O
B
A
▲
Figure 2. Exploration of the desired linker structure with a satisfactory spatial arrangement and
an optimal distance between unit (A) and unit (B).
The synthetic routes used in this work are summarized in Schemes 1, 2, 3, 4, 5, 6 and 7.
Scheme 1. a Synthesis of compound 6, which has a trans-olefin chain as a linker between the N-
(2,6-diisopropylphenyl)acetamide unit (A) and 2-(methylthio)benzo[d]oxazole unit (B).
O
i
H
H2N
SH
N
+
Cl
N
O
3
5
4
ii
H
O
N
N
S
O
6
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a Reagents and conditions: i) (E)-6-chloro-2-hexenoyl chloride, Et3N, CHCl3, rt, 46%; ii) 2-
mercaptobenzo[d]oxazole (5), K2CO3, 18-crown-6, DMF, 80 °C then 100 °C, 30%.
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(E)-6-Chloro-2-hexenoyl chloride was obtained as described in the literature 36 and subsequently
used for acylation of 2,6-diisopropylaniline (3) to yield anilide 4. The conversion to thioether 6
was achieved by reacting 4 with 5 in the presence of a base and a phase-transfer catalyst.
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Scheme 2. a Synthesis of compound 9, which has a benzene ring as a linker between the N-(2,6-
diisopropylphenyl)acetamide unit (A) and 2-(methylthio)benzo[d]oxazole unit (B).
i,ii
iii
H
H
O
N
OH
N
N
Br
Br
S
O
O
O
8
9
7
a Reagents and conditions: i) (COCl)2, cat. DMF, CHCl3, rt; ii) 3, Et3N, CHCl3, rt, 20% over 2
steps iii) 5, K2CO3, 18-crown-6, DMF, 80 °C, 51%.
3-Bromomethylphenylacetic acid (7) was prepared from 2-(m-tolyl)acetic acid as described in
the literature. 37 The acylation of 3 with 7 yielded anilide 8, which was subsequently converted to
thioether 9 as described in Scheme 1.
Scheme 3. a Synthesis of compound 22, which has a cis-cyclohexane ring as a linker between the
N-(2,6-diisopropylphenyl)acetamide unit (A) and 2-(methylthio)benzo[d]oxazole unit (B).
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O
O
O
i
ii
iii
HOOC
COOEt
t-BuPh2SiO
COOEt
HO
COOEt
iv
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COOEt
COOEt
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t-BuPh2SiO
t-BuPh2SiO
t-BuPh2SiO
t-BuPh2SiO
OH
O
v
vi
vii
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17
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O
N
Br
HO
S
O
O
O
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x, xi
xii
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OEt
OEt
OH
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20
xiii
O
N
O
N
S
S
O
O
Cl
N
H
21
22
a Reagents and conditions: i) EtOH, reflux, 39%; ii) Et3N, ClCOOEt, THF, 0 °C and then NaBH4
/H2O, THF, 0 °C, 51%; iii) t-Bu(Ph)2SiCl, imidazole, DMF, rt, 73%; iv) DIBAL, toluene, -78 °C,
96%; v) Et3N, PySO3, DMSO, 0 °C; vi) NaH, (EtO)2P(O)CH2COOEt, THF, 0 °C then -78 °C,
89% over 2 steps; vii) H2, Pd/C, EtOH, rt, 77%; viii) TBAF, THF, rt, 97%; ix) Ph3P, NBS,
CH2Cl2, rt, 78%; x) 5, K2CO3, 18-crown-6, DMF, 80 °C, 89%; xi) 2 N NaOH, EtOH, 55 °C,
98%; xii) (COCl)2, cat. DMF, CHCl3, rt; xiii) 3, Et3N, CHCl3, rt, 42% over 2 steps.
Solvolysis of cis-4-cyclohexene-1,2-dicarboxylic anhydride (10) in EtOH yielded the ring-
opened ester 11. Treatment of 11 with ethyl chloroformate and Et3N in THF afforded the
intermediate mixed anhydride, followed by reduction with NaBH4 for conversion to alcohol 12.
Alcohol 12 was protected with tert-butyldiphenylchlorosilane in the presence of imidazole in
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DMF to afford the TBDPS ether 13. Reduction of 13 with DIBAL yielded alcohol 14. Exposure
of 14 to the sulfur trioxide-pyridine complex in DMSO yielded aldehyde 15, which was treated
with a phosphonate ylide prepared from NaH and triethyl phosphonoacetate to obtain acrylate 16.
Hydrogenation of 16 in the presence of Pd/C under a hydrogen atmosphere gave 17, which was
subjected to desilylation with TBAF to obtain alcohol 18. Treatment of 18 with NBS and Ph3P
afforded bromo compound 19. Reaction of 19 with 5 in the presence of K2CO3 and 18-crown-6
yielded a thioether, which was subsequently hydrolyzed by 2 N NaOH in EtOH to afford
carboxylic acid 20. Treatment of 20 with oxalyl chloride in the presence of a catalytic amount of
DMF in CHCl3 gave acid chloride 21. The N-acylation reaction between 3 and 21 under basic
conditions provided the desired anilide 22.
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Scheme 4. a Synthesis of compound 33, which has a trans-cyclohexane ring as a linker between
the N-(2,6-diisopropylphenyl)acetamide unit (A) and 2-(methylthio)benzo[d]oxazole unit (B).
OH
OBn
OBn
OBn
OBn
i)
iv)
ii)
iii)
OH
OMEM
OH
O
O
OH
O
23
24
25
27
26
Br
Br
Br
OH
vi)
vii)
v)
viii)
OH
Cl
O
OMEM
OMEM
O
O
O
28
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O
N
S
Br
ix)
x)
H
H
N
N
O
O
32
33
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a Reagents and conditions: i) NaH, 15-crown-5, BnBr, DMF, 0 °C then rt, 45%; ii) Et3N, PySO3,
DMSO, 0 °C; iii) Na2HPO4, 2-methyl-2-butene, NaClO2, t-BuOH, H2O, rt, 65% over 2 steps; iv)
i-Pr2EtN, MEMCl, CH2Cl2, 0 °C, 68%; v) H2, Pd/C, EtOH, rt, 97%; vi) Ph3P, NBS, CH2Cl2, rt,
43%; vii) 3 N HCl, THF, 40 °C, 85%; viii) (COCl)2, cat. DMF, CHCl3, rt; ix) 3, Et3N, CHCl3, rt,
32% over 2 steps; x) 5, K2CO3, 18-crown-6, DMF, 80 °C, 92%.
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(trans-(4-(Benzyloxy)methyl)cyclohexyl)methanol (24) was prepared from trans-1,4-
38
cyclohexanedimethanol (23) according to the method described in a patent application.
Exposure of 24 to the sulfur trioxide-pyridine complex in DMSO yielded aldehyde 25, which
was oxidized to carboxylic acid 26 by sodium chlorite. Protection of 26 with MEMCl in the
presence of Hünig’s base yielded MEM ester 27. Hydrogenolysis of 27 in the presence of Pd/C
under a hydrogen atmosphere yielded alcohol 28. Treatment of 28 with NBS and Ph3P afforded
bromo compound 29, which was deprotected under acidic condition to yield carboxylic acid 30.
Treatment of 30 with oxalyl chloride via a conventional procedure yielded acid chloride 31. The
N-acylation reaction between 3 and 31 under basic conditions yielded anilide 32. The desired
thioether 33 was obtained by the S-alkylation reaction of 5 with 32 under the conditions
described above.
Scheme 5. a Synthesis of compound 38, which has a 1,4-diazacycloheptane ring as a linker
between the N-(2,6-diisopropylphenyl)acetamide unit (A) and 2-(methylthio)benzo[d]oxazole
unit (B).
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H
ii
i
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H
N
NH
N
+
Br
N
HO
N
HO
O
O
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iii
H
N
H
N
O
N
N
N
MsO
N
S
O
O
N
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a Reagents and conditions: i) K2CO3, MeCN, rt, 87%; ii) Et3N, DMAP, MsCl, THF, 0 °C; iii) 5,
K2CO3, 18-crown-6, DMF, 80 °C, 81% over 2 steps.
The N-alkylation reaction between 3-(1,4-diazepan-1-yl)propan-1-ol (34) and 2-bromo-N-(2,6-
diisopropylphenyl)acetamide (35) 32 provided anilide 36, which was converted to mesylate 37
under the reaction conditions described above. The desired thioether 38 was obtained by reacting
37 with 5 in the conventional manner described above.
Scheme 6. a Synthesis of compound 42, which has a 1,4-diazacyclohexane ring as a linker
between the N-(2,6-diisopropylphenyl)acetamide unit (A) and 2-(methylthio)benzo[d]oxazole
unit (B).
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i
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N
NH
35
+
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HO
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HO
N
O
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H
N
iii
H
N
N
N
O
N
MsO
N
O
S
N
O
41
42
a Reagents and conditions: i) K2CO3, MeCN, 80 °C, 63%; ii) Et3N, DMAP, MsCl, THF, 0 °C; iii)
5, K2CO3, 18-crown-6, DMF, 80 °C, 60% over 2 steps.
The N-alkylation reaction between 3-(piperazin-1-yl)propan-1-ol (39) and 35 yielded the
corresponding alcohol 40, which was converted to mesylate 41 under the reaction conditions
described above. The desired thioether 42 was obtained by reacting 41 with 5 under the reaction
conditions described above.
a
Scheme 7. Synthesis of compounds from 55 to 65, with a 1,4-diazacyclohexane ring (lacking
only one methylene unit) as a linker between a N-phenylacetamide or N-pyridylacetamide unit
(A) and a 2-(methylthio)benz[d]azole or 2-(methylthio)oxazolo[4,5-b]pyridine unit (B).
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R1
R1
R1
i
H
H
N
R2
N
R2
NH
+
Br
N
N
X
X
HO
R1
O
N
O
7
HO
8
9
1
2
1
2
46
47
48
R = i-Pr, R = H, X = CH
43
35
44
45
R = i-Pr, R = H, X = CH
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1
2
1
2
R = R = i-Pr,
X = CH
R = R = i-Pr,
X = CH
1
2
1
2
R = SMe, R = Me, X = N
R = SMe, R = Me, X = N
R1
H
ii
iii
Y
N
N
R2
SH
+
N
X
Z
R1
N
O
MsO
1
2
5 Y = O, Z = CH
49
50
51
R = i-Pr, R = H, X = CH
52
53
54
Y = NH, Z = CH
Y = S, Z = CH
Y = O, Z = N
1
2
R = R = i-Pr,
X = CH
1
2
R = SMe, R = Me, X = N
1
2
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56
57
58
59
60
61
62
63
2
R = i-Pr, R = H, X = Z = CH, Y = O,
1
2
R1
H
R = i-Pr, R = H, X = Z = CH, Y = NH,
1
2
R = i-Pr, R = H, X = Z = CH, Y = S,
R2
1
2
N
R = i-Pr, R = H, X = CH,
Y = O, Z = N
N
1
2
X
R = R = i-Pr,
X = Z = CH, Y = O,
Y
R1
1
2
N
O
S
R = R = i-Pr,
X = Z = CH, Y = NH,
X = Z = CH, Y = S,
1
2
N
Z
R = R = i-Pr,
1
2
R = R = i-Pr,
X = CH,
Y = O, Z = N
1
2
R = SMe, R = Me, X = N,
R1 = SMe, R2 = Me, X = N,
Y = O, Z = CH
Y = NH, Z = CH
Y = S, Z = CH
1
2
64
65
R = SMe, R = Me, X = N,
1
2
R = SMe, R = Me, X = Z = N, Y = O,
a Reagents and conditions: i) K2CO3, MeCN, 80 °C, 80−97% ; ii) Et3N, DMAP, MsCl, THF,
0 °C; iii) 5, 52, 53 or 54, K2CO3, 18-crown-6, DMF, 80 °C, 41−88% over 2 steps.
Similar to the reaction described above, the N-alkylation reaction between 3-(piperazin-1-
yl)ethan-1-ol (43) and bromo compounds 35, 44 and 45 yielded the corresponding alcohols 46,
47 and 48, which were converted to mesylates 49, 50 and 51 under the reaction conditions
described above. The desired thioethers from 55 to 65 were obtained by reacting mesylates 49,
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50 and 51 with 2-mercaptobenzo[d]oxazole (5), 2-mercaptobenz[d]-imidazole (52), 2-
mercaptobenzo[d]thiazole (53) and 2-mercaptooxazolo[4,5-b]pyridine (54) under the reaction
conditions described above.
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60
Results and Discussion
The prepared compounds were evaluated for in vitro ACAT inhibition according to a modified
version of the method described by Heider et al. 39 by using two enzyme sources: rabbit aorta
homogenate and rabbit small intestine microsomes. The J774 macrophage cell culture assay was
40
performed to assess the inhibition of the target cells by systemic antiatherosclerotic agents.
ACAT inhibition by all the prepared compounds is described herein and compared with that by
32
our previously reported compound 1
as an improvement index. The relationships between
ACAT inhibition (IC50) and the linker structure are summarized in Table 1. Based on compound
1 with the flexible linker, we investigated the impact of the intestinal IC50/aortic IC50 selectivity
ratio when the linker structure was slightly modified. Compound 6, containing (E)-olefin,
maintained almost a 5-fold selectivity whereas compound 9, containing a benzene ring, lost
selectivity. Interestingly, compound 33, containing a spatially arranged trans-cyclohexane ring,
exhibited a 9.6-fold increase in selectivity, whereas compound 22, containing a cis-cyclohexane
ring, was at 3.9-fold selective. It has been suggested that the spatially arranged trans-orientation
in head-to-tail molecules might lead to low binding affinity with the intestinal ACAT and to
weakening of intestinal ACAT inhibition, thereby enhancing the intestinal IC50/aortic IC50
selectivity ratio.
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Table 1. Effects of the spatial structure and arrangement of the linker between N-2,6-
diisopropylaniline and 2-thiobenzo[d]oxazole on ACAT inhibition.
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8
9
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43
44
45
46
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48
49
50
51
52
53
54
55
56
57
58
59
60
O
N
H
Linker
N
S
Aortic
Intestinal
J774 Cells
Intestinal/
Aortic Ratio
Compound
Linker Structure
cLogP d
7.25
IC50 (M)
IC50 (M) b
IC50 (M) c
a
1
0.004
0.036
0.021
0.180
5.3
5.0
0.007
2.7
O
6
9
5.39
5.73
O
0.05
0.051
0.093
1.0
3.9
1.6
O
22
33
0.024
0.37
7.24
6.71
O
0.026
0.250
9.6
1.2
O
38
42
0.025
0.019
0.064
0.039
2.6
2.1
0.43
0.25
4.72
4.15
N
N
O
N
N
O
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55
0.012
0.037
3.1
0.26
3.82
N
N
O
7
8
9
a
b
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33
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46
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49
50
51
52
53
54
55
56
57
58
59
60
IC50 (M) against rabbit aortic ACAT activity. IC50 (M) against rabbit intestinal
microsome ACAT activity. c IC50 (M) against esterified cholesterol accumulation in J774 cells.
d cLogP was calculated via ChemDraw/Pro 15.1.
Therefore, we envisioned the use of the spatial trans-orientation in our molecular design. Next,
we focused on the role of the basic amine residue in order to improve aqueous solubility in acidic
media, as shown in the following examples.
O
N
N
N
H
N
N
N
H
H
2HCl
N
N
(HCl)
H
N
O
O
PD-132301
cLogP = 6.53
Solubility 2.18 mg/mL (pH 1.0)
PD-132301-2
YM-17E
cLogP = 8.70
Solubility 10 mg/mL (0.025-N HCl) lit. 42
(HCl)
Solubility 6.4 mg/mL (0.1-N HCl) lit. 41
N
N
H
H
O
O
N
N
S
S
N
N
O
O
66
67
cLogP = 5.50
cLogP = 6.24
Solubility 0.18 mg/mL (pH 1.2)
IC50 = 0.036 M (aorta)
IC50 = 0.077 M (intestine)
IC50 = 0.820 M (J774)
Solubility 0.4 mg/mL (pH 1.2)
IC50 = 0.096 M (aorta)
IC50 = 0.130 M (intestine)
IC50 = 5.60 M (J774)
Figure 3. Physical and biological properties of ACAT inhibitors containing a N,N-
dimethylamino group and a N,N-dimethylaminomethyl group
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As shown in Figure 3, introduction of the N,N-dimethylamino group into molecules such as 1-
5
6
(2,6-diisopropylphenyl)-3-((1-(4-(dimethylamino)benzyl)cyclopentyl)methyl)urea (PD-132301)
7
8
9
41
and 1,1'-(1,3-phenylenebis(methylene))bis(1-cycloheptyl-3-(4-(dimethylamino)phenyl)urea)
42
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dihydrochloride (YM-17E) has been very effective at increasing aqueous solubility in acidic
media. Comparison of compounds 66 and 67 32 shows that the N,N-dimethylaminomethyl group
also contribute to doubling the aqueous solubility (from 0.18 to 0.4 mg/mL) by increasing the
basicity but dramatically decreased ACAT inhibition in J774 cells. As reported previously, 32 the
polar-ionic interaction around the binding site not only negatively affected the adaptability of the
ligand-binding pocket or cleft in our head-to-tail design but also potentially obstructed the
molecular permeability of the J774 cell membrane. Accordingly, this insight led us to
incorporate two nitrogen atoms into the linker. Compound 38 (with a homopiperazine linker) had
a ca. 2 orders of magnitude lower cLogP value than that of 33 (with a trans-cyclohexane linker)
but successfully maintained the inhibitory activity toward aortic ACAT. Notably, 42 and 55
(with piperazine linkers lacking one or two methylene units) exhibited slightly more potent
inhibitory activity toward intestinal ACAT and J774 cells than 38. Having succeeded in
minimizing the value of cLogP from 4.72 to 3.82, we next investigated the appropriate
combination of substituent groups (R1 = R2 and/or R3) and atoms (X, Y, Z) based on the template
structure of 55. The ACAT inhibitory activities of compounds 55 to 65 are summarized in Table
2.
Table 2. Effects of the substituent groups and incorporated hetero atoms at each position on the
1,3-benzazole moiety (Y = O, NH, or S; Z = CH or N) and arylacetamide moiety (X = CH or N
and substituent groups R1and R2) on ACAT inhibition.
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R1
R1
H
N
R2
N
X
Y
N
N
O
S
Z
9
10
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49
50
51
52
53
54
55
56
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58
59
60
Aortic
Intestinal
J774 Cells
Intestinal/
Aortic Ratio
R1
R2
Y
Z
Compound
X
cLogP d
IC50 (M) a IC50 (M) b
IC50 (M) c
55
56
57
58
59
60
61
62
63
2
i-Pr
H
H
H
H
CH
O
CH
0.012
0.11
0.037
0.51
0.036
0.32
0.270
0.41
0.31
1.3
3.1
4.6
2.3
2.9
7.9
1.8
2.4
4.3
0.9
5.4
0.9
1.8
0.26
0.28
0.17
0.45
0.25
0.49
0.41
0.37
0.009
0.026
0.003
0.1
3.82
3.97
4.49
2.77
5.24
5.39
5.92
4.19
4.07
4.22
4.78
3.02
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
CH NH CH
CH
CH
S
O
O
CH
N
0.016
0.11
i-Pr CH
CH
0.034
0.23
i-Pr CH NH CH
i-Pr CH
i-Pr CH
S
O
O
CH
N
0.13
0.30
SMe
SMe
SMe
SMe
Me
Me
Me
Me
N
N
N
N
CH
0.033
0.084
0.041
0.12
0.029
0.45
0.038
0.21
NH CH
64
65
S
CH
N
O
a
b
IC50 (M) against rabbit aortic ACAT activity. IC50 (M) against rabbit intestinal
microsome ACAT activity. c IC50 (M) against esterified cholesterol accumulation in J774 cells.
d cLogP was calculated by ChemDraw/Pro 15.1.
For selection of precandidate aqueous-soluble ACAT inhibitors, we set the threshold value for
ACAT inhibitory activity (M) against J774 cells as 0.1 and that for cLogP as 4.3, which were
low and reasonably achievable. Finally, we selected 2, 63 and 65 and evaluated the lipid-
modulating abilities (CE /free cholesterol (FC) ratio) of these compounds in each organ, which
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would be ultimately the same output as that for the treatment of incurable diseases that involve
ACAT-1 overexpression. The evaluation was performed with a diet-induced atherosclerosis
model using Bio F1B hamsters as described previously. 32 To investigate potential drug exposure,
we conducted a PK study using precandidates 2, 63 and 65, which were orally administered to
nonfasted male rats at a dose of 30 mg/kg.
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a
Table 3. Comparison of the physical and PK properties of precandidates 2, 63 and 65 with
those of the lead compound 1 under nonfasted conditions b.
Compound
1 c
2
63
65
Solubility
at pH 1.2
19 mg/mL
17 mg/mL
33 mg/mL
0.03 g/mL
Tmax
Cmax
T1/2
3.2 h
107 ng/mL
ND d
0.7 h
192 ng/mL
2.1 h
2.8 h
158 ng/mL
10.1 h
0.5 h
1038 ng/mL
3.4 h
AUCinf
358 ng·h/mL
594 ng·h/mL
4125 ng·h/mL
2825 ng·h/mL
a
b
The values are the means of > 3 experiments ± standard deviations (SDs). A 30 mg/kg dose
(1) in 0.5% MC suspension solution and (2, 63 and 65) in 0.05 N HCl solution were orally
c
administered to male rats (n = 3) under nonfasted conditions. The data shown for compound 1
was cited from our previous paper.32 d ND (not determined) because the drug concentration for
one rat continued to increase 6 h after administration, while that for the other two rats remained
stable.
As shown in Table 3, the compounds with the piperazine linker, namely, 2, 63 and 65, exhibited
dramatically enhanced aqueous solubilities, with values of 19, 17 and 33 mg/mL, respectively.
These major improvements led to an 11.5- and 7.9-fold increase in the drug exposure (AUCinf) of
63 and 65, respectively, compared with that of 1. To determine the doses of the three compounds,
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we considered the weakest activity (0.1 M) of 65 against J774 macrophages and determined the
doses of 2 and 63 (1, 3, 10 and 30 mg/kg) and those of 65 (3, 10, 30 and 100 mg/kg) for the
dietary drug administration study, which utilized Bio F1B hamsters fed a diet that was high in fat
and cholesterol for 10 weeks.
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IC50 = 31 M (rabbit aorta homogenate) 32
IC50 = 13 M (rabbit intestinal microsomes)
O
O
O
In the cholesterol-fed F1B hamster model 43
S
N
H
O
Reduction of lipid accumulation area with decreasing plasma TC
68% at a dose of 3 mg/kg
86% at a dose of 10 mg/kg
CI-1011
93% at a dose of 30 mg/kg
Figure 4. Structure and pharmacological properties of CI-1011
43-49
2,6-Diisopropylphenyl(2-(2,4,6-triisopropylphenyl)acetyl)sulfamate (CI-1011, Avasimibe)
demonstrated hypolipidemic and antiatherosclerotic effects in several animal models and was in
phase III clinical trials. We used this compound as reference compound for in vivo tests.
The nonselective ACAT inhibitor 63 at doses of 3, 10 and 30 mg/kg significantly decreased the
lipid accumulation areas in the aortic arch (37, 32 and 28% relative to the control, respectively)
along with reducing the total cholesterol (TC) level in the plasma (73, 28 and 24% relative to the
control, respectively). The pharmacological behavior of 63 was similar to that of CI-1011.
Interestingly, the weakest ACAT inhibitor, 65, even at doses of 3 and 10 mg/kg, markedly
decreased the lipid accumulation areas (49 and 34% relative to the control, respectively) without
decreasing the plasma TC level, as shown in Figure 5. The potent inhibitor 2, with a higher
selectivity toward aortic ACAT than 65, showed only moderate effects on the lipid accumulation
areas (71 and 53% relative to the control, respectively) at the same doses. Next, we asked why 65
had a strong effect on the aortic ACAT. The PK profile indicated that 65 had a high Cmax (1038
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ng/mL), a short Tmax (0.5 h) and a rather large AUCinf (2825 ng·h/mL) with a T1/2 of 3.4 h,
while 2 exhibited a low Cmax (192 ng/mL) and a short Tmax (0.7 h) but a moderate AUCinf (594
ng·h/mL) with a short T1/2 (2.1 h). Based on the ACAT inhibitory activities of both compounds
(2: IC50 = 0.026 M vs 65: IC50 = 0.1 M) toward J774 cell macrophages (i.e., antifoaming
action), the effective capacity (F) of ACAT inhibitors could be expressed as an index by the
following equation: F = Cmax/MW/IC50. The F value of 2 was calculated to be 14.7, whereas
that of 65 was 20.6. Accordingly, the effective capacity of 2 was ca. 70% as much as that of 65.
The actual lipid accumulation areas of 65 at doses of 3 and 10 mg/kg (49 and 34% relative to the
control, respectively) were consistent with the calculated areas for 2 (71 × 0.7 = 50 and 53 × 0.7
= 37% relative to the control, respectively).
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Figure 5. Effects of CI-1011, 2, 63 and 65 on the plasma TC levels and lipid accumulation areas.
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Compounds 2 and 63 were administered at doses of 1, 3, 10, 30 mg/kg, whereas 65 was
administered at doses of 3, 10, 30, 100 mg/kg, via the diet to Bio F1B hamsters fed a diet that
was high in fat and cholesterol. The control compound CI-1011 was used at the same dose
32
setting (0.1, 1, 3, 10 mg/kg) that was described previously. The % value ± SD was calculated
7
relative to the control. * p<0.05 and ** p<0.01 vs. control; statistical analysis was conducted
using Dunnett’s test.
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As shown, the F value allowed estimation of both pharmacological effects. On the other hand,
the nonselective ACAT inhibitor 63 exhibited a low Cmax (158 ng/mL) with a medium Tmax
(2.8 h), but exhibited the largest AUCinf (4125 ng·h/mL) with a long T1/2 (10.1 h). This
compound acted on the liver and intestinal ACAT over a long duration and contributed a
significant decrease in the plasma TC levels for the suppression of lipid accumulation areas.
Therefore, the pharmacodynamics (PD) of aortic ACAT might be more significantly affected by
a large Cmax than by a large AUCinf. In fact, a transient exposure of aortic ACAT to a large
amount of drug might prolong the duration of the inhibitory effect on aortic ACAT. In other
words, the inhibition of aortic ACAT seems to be different from that of other enzymes, such as
HMG-CoA reductase. 50-53 To further elucidate the lipid-modulating potential of 2, 63 and 65 in
each organ, we measured the CE/FC ratio in the plasma, liver, intestine and adrenal gland. The
relative relationships of the ratio of the change in lipid accumulation areas and plasma TC levels
and the lipid proportion ratio (CE/FC) in the liver, intestine and adrenal gland with the dose
responses are presented as scatter plot graphs in Figure 6.
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Figure 6. Relationships of the relative ratios of lipid accumulation areas and plasma TC levels
and the lipid proportion ratio (CE/FC) in the liver, intestine and adrenal gland with the dose
responses.
In the scatter plot graphs, the pharmacological profile of the ACAT inhibitors precisely
indicated the degree of the effect on each organ. As nonselective ACAT inhibitors, the slopes of
all the lines for 63 were similar to those for CI-1011. In contrast, both inhibitors 2 and 65
exhibited large discrepancies between the plasma TC lines and the lipid accumulation area lines,
which were positioned the lowest. This important finding reveals aortic-selective ACAT
inhibition in vivo. Notably, the adrenal gland CE/FC lines for CI-1011, 2 and 65 were positioned
the highest, whereas that for 63 was the lowest. CI-1011 has actually been reported to have a low
adrenal effect. 54 This behavior was consistent with the results of our preliminary adrenal toxicity
test after 24 h when single intravenous injection of 63 or 65 were administered to male rabbits.
This test had the advantage of predicting adrenal toxicity in a sensitive and prompt manner, 55 in
contrast to the toxicity test that involved repeated oral administration over a long period. While
63 induced the necrosis of adrenal zona fasciculata cells in three of four rabbits, even at a dose of
0.1 mg/kg, 65 exhibited similar toxicity in one of four rabbits at a dose of 5 mg/kg. The toxicity
level of 63 was revealed to be the same as that of PD-132301-2, which was a positive control. 56-
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The no-observed-adverse-effect level (NOAEL) of CI-1011 was determined to be 5 mg/kg.
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Accordingly, the adrenal gland CE/FC line can be regarded as a safety index for adrenal toxicity.
At this stage, 65 was the most attractive aortic-selective ACAT inhibitor with good exposure and
safety. For further profiling of 2, 63 and 65 before selection of a clinical candidate, we assessed
the compounds via the full Ames test (TA98, TA100, TA1535, TA1537, WPuvrA) and the
chromosomal aberration test. Although the three precandidates passed the Ames test, the
compounds 63 and 65 tested positive in the chromosomal aberration test (Supporting
Information). In the three compounds 2, 63 and 65, N-(6-methyl-2,4-bis(methylthio)pyridin-3-
yl)acetamide was a common structure in the head moiety. We concluded that the potential
genotoxicity originated from the structure of the 2-thiobenzo[d]oxazole and 2-thiooxazolo[4,5-
b]pyridine group in the tail moiety. Unexpectedly, both compounds 63 and 65 were identified as
unstable substrates upon exposure to acidic medium in the chemical stability test (Supporting
Information). Therefore, we abandoned the development of 63 and 65 as active pharmaceutical
ingredients (APIs). Next, we focused our efforts on further pharmacological studies of 2. As
previously reported, we verified the isoform selectivity of 2 using human ACAT-1 and ACAT-2
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and identified this compound to be a potent ACAT-1 inhibitor with 229-fold selectivity for
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ACAT-2.
Having investigated the reason underlying the large discrepancy in the value of
isoform selectivity between human ACATs and rabbit ACATs in an ACAT assay system, we
recently reported that the binding of the plasma protein with the substrate is crucial for the
measurement of ACAT inhibition in the conventional assay. According to the reported
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simulation, the aortic IC50 value (84 nM) of 2 decreased to 2.3 nM when using the rabbit
unbound plasma fraction (fp) (2.7%) (Supporting Information). Due to plasma protein binding,
the IC50 of aortic ACAT inhibition was weakened from approximately 2.3 nM to 84 nM. On the
other hand, the intestinal ACAT inhibition (450 nM) in the digestive tract was not affected;
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therefore, the value of isoform selectivity was identified to be approximately 196 (450 divided
by 2.3 is 195.6). 60 This simulation result is consistent with human ACAT isoform selectivity and
accounts for the obtained pharmacological behavior in vivo. In the precise evaluation of F1B
hamsters on a high-fat diet, the suppression of lipid accumulation areas could be significantly
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compensated even at a dose of ≥1 mg/kg 2 without affecting the plasma TC levels when dietary
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administration was replaced with oral administration.
We interpreted that the reduction in
dosing was attributed to a ca. 3-fold increase in drug exposure by switching to oral drug
administration from dietary administration; the data showed that the Cmax was 568 ng/mL when
the drug was orally administered to fasted male rats at a dose of 30 mg/kg. Furthermore, when
apoE-knockout mice were treated for 12 weeks with a dose of 60 mg/kg, we observed the direct
plaque-modulating effects of 2 on the vascular wall independently of the plasma TC levels. 31 In
the event of acute coronary syndrome (ACS), vulnerable plaques are characterized by
accumulation of macrophage-derived foam cells and thin fibrous caps, while stable plaques are
characterized by collagen-rich fibrous caps with fewer macrophages than vulnerable plaques.
Rupture of the fibrous cap may occur, thereby exposing highly procoagulant material to the
circulating blood, leading to occlusive thrombus formation and subsequent ischemia or
infarction. 61 Therefore, our final therapeutic goal is to transform vulnerable plaque into stable
plaque for the prevention of plaque rupture. Having observed a potentially therapeutic effect in
the prevention of ACS, we next conducted a PK study of 2 when orally administered to fasted
and nonfasted dogs for extrapolation of human PK.
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a
Table 4. Comparison of the pharmacokinetic properties of 2 under fasting and nonfasting
conditions b.
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Condition
Tmax (h)
Cmax (ng/mL)
Area under the curve
(AUC) 0-8 h (ng·h/mL)
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Nonfasted
Fasted
2.3 ± 1.3
1.0 ± 0.7
1424 ± 1649
5466 ± 4010
3575 ± 4012
13932 ± 7825
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a Data are shown as the means ± SDs. b A 10 mg/kg dose in capsules was orally administered to
male dogs (n = 4) under fasting and nonfasting conditions.
As shown in Table 4, interestingly, the Cmax and AUCinf values for the aqueous-soluble
compound 2 under fasting conditions were 3.8-fold higher than those under nonfasting
conditions. This finding is a positive sign in terms of safety test. Because the NOAEL of the
highly lipophilic compound 1 could not be determined, saturation of drug exposure was reached
due to poor absorption in male dogs at doses of 30 and 100 mg/kg over 2 weeks. The
improvement of drug exposure between lead compound 1 and candidate compound 2 is
summarized in Figure 7. The marked enhancement of aqueous solubility (up to 19 mg/mL at pH
1.2) led to dramatic improvement of oral absorption (the Cmax of 2 was 1100-fold higher than
that of 1 in fasted dogs).
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a
Figure 7. Plasma drug concentration of 1 at a dose of 30 mg/kg and 2 at a dose of 10 mg/kg
after a single oral administration to male dogs (n = 3 and n = 4) under nonfasting or fasting
conditions. a The data for compound 1 was cited from our previous paper. 32
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In safety tests for 2, the NOAELs were determined to be 10 mg/kg in both dogs (4 weeks) and
monkeys (26 weeks) without sex discrimination. At the beginning of this study, we were very
concerned about whether the adrenal toxicity could be avoided. To investigate this issue, the
drug tolerance of a healthy volunteer in a phase 1 study far surpassed the safety threshold (Cmax,
1 g/mL, AUC, 3 g·h/mL) observed in the safety test for monkeys. In a phase II study, the
clinical candidate 2 showed stable linear exposure in terms of Cmax and AUC between the low-
and high-dose groups for 26 weeks. In a clinical study on the prevention and early intervention
of cardiovascular disease, the lipid profiles of patients should be well controlled to decrease
plasma TC levels to 171 mg/dL and low-density lipoprotein cholesterol (LDL-C) levels to 101
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mg/dL by statin. We stated that a so-called add-on therapy with statin would hinder the
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assessment of the capability of ACAT inhibitors. Based on our experience,
we wondered
whether it would be appropriate to evaluate the efficacy of ACAT inhibitors under circumstances
where ACAT activity would be markedly downregulated. We believe that it would be beneficial
for research to be focused on diseases that involve ACAT-1 overexpression, such as aggressive
cancers and Alzheimer’s disease, which is independent of cholesterol metabolism in the brain.
Conclusion
Starting from the highly lipophilic aortic ACAT inhibitor 1, we used a solubility-driven
structural optimization and inserted a piperazine unit in the place of a 6-methylene chain into the
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linker region. By optimization and selection, we have overcome several drawbacks associated
with 1 and succeeded in obtaining the marked improvement of the aqueous solubility (up to 19
mg/mL at pH 1.2) and oral absorption (the Cmax of 2 was 1100-fold higher than that of 1 in
fasted dogs) of 2 compared with those of 1. We also serendipitously discovered 2 to be a potent
inhibitor of human ACAT-1, with 229-fold selectivity for this protein over human ACAT-2, and
demonstrated the unique pharmacological effects of this compound in plaque stabilization. After
this compound passed safety tests, we designated 2 as a clinical candidate named K-604, which
was found to be well tolerated in a phase 1 study and maintained stable exposure over 26 weeks
in a phase 2 clinical study. Rather than the treatment of atherosclerosis, we believe that K-604
has potential therapeutic applications for the treatment of incurable diseases that involve ACAT-
1 overexpression. Recently, we succeeded in establishing an intranasal administration method for
efficient delivery of K-604 into the brain, which made it possible to substantially modulate brain
cholesterol levels in a smooth manner despite a short duration of exposure. We will report these
results in the near future.
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Experimental Section
All procedures performed on animals in this study were 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 active compounds for PAINS and found that they were negative.
Chemistry.
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Commercially available reagents and solvents were used without further purification. Thin-
layer chromatography (TLC) analyses were conducted using silica gel 60 F254 plates (Merck).
1H-nuclear magnetic resonance (NMR) spectra were recorded using a JEOL JNM-LA 400 MHz
and JEOL GSX-270. 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. Infrared (IR) spectra were recorded using a JASCO ALOR-III. Mass spectra
were obtained on a JEOL MS-BU20 mass spectrometer (MS). Low-resolution mass spectra were
recorded on a JEOL JMS-D-300 MS. Elemental analyses (C, H, and N) were performed using
Yanaco MT-5. Melting points were determined in open glass capillaries using a Buchi B-545
melting point apparatus. Purities of all test compounds were greater than 95% based on HPLC
chromatogram on a Shimadzu LC-20A series. LC/MS system (Shimadzu LC-10A and AB Sciex
API 165) were used for the measurement of plasma drug concentration.
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Materials
3-(1,4-Diazepan-1-yl)propan-1-ol, 3-(piperazin-1-yl)propan-1-ol and 2-mercaptooxazolo[4,5-
b]pyridine were commercially available.
The syntheses of the target compounds 2, 63, and 65 are described below. Detailed
experimental procedures for the syntheses of all other compounds, their intermediates and the
characterization data are provided in the Supporting Information.
Representative synthetic procedure for compounds 2, 63 and 65.
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2-[4-[2-(Benzimidazole-2-ylthio)ethyl]piperazin-1-yl]-N-(2,4-bis(methylthio)-6-methyl-3-
pyridyl)acetamide (the free amine of 2). 6-Methyl-2,4-bis(methylthio)pyridin-3-amine was
obtained according to a method described in a patent (WO9104027, Sep 15, 1989). Step 1. A
solution of bromo acetylbromide (62.7 g, 0.311 mol) in CH2Cl2 (200 mL) was added dropwise
to a stirred solution of 6-methyl-2,4-bis(methylthio)pyridin-3-amine (60.1 g, 0.30 mol) and N,N,-
dimethylaniline (47.9 g, 0.395 mol) in CH2Cl2 (300 mL) at 0 °C over 1 h. The reaction mixture
was allowed to stand at 0 °C for 12 h. The precipitated salt was filtered out and washed with cold
EtOH (300 mL) and Et2O (300 mL) to yield 2-bromo-N-(6-methyl-2,4-bis(methylthio)-pyridin-
3-yl)acetamide (45) (64.2 g, 67%) as colorless fine needles. The combined filtrates (mother
liquors) were concentrated under reduced pressure. The residue was dissolved in CHCl3 (100
mL) and crystalized from Et2O (200 mL) to yield 45 (12.37 g, 13 %) as colorless powdery
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crystals. Mp 157-158 ℃; IR (KBr) cm-1: 3222, 3184, 3042, 2999, 1668, 1565, 1522; H-NMR
(CDCl3) : 2.43 (3H, s), 2.50 (3H, s), 2.53 (3H, s), 4.07 (2H, s), 6.67 (1H, s), 7.58 (1H, br s);
Anal. Calcd for C10H13BrN2OS2: C, 37.39; H, 4.08; N, 8.72; Br, 24.87; S, 19.96. Found: C,
37.41; H, 4.08; N, 8.59; Br, 24.99; S, 20.13. Step 2. K2CO3 (37.5 g, 0.271 mol) was added to a
solution of 1-(2-hydroxyethyl)piperazine (43) (35.3 g, 0.271 mol) and 45 (64.2 g, 0.20 mol) in
MeCN (1.2 L) under ice-cold conditions. The reaction mixture was allowed to warm to rt,
stirred for 23 h, and then diluted with water (1.0 L) and CHCl3 (1.5 L). The organic layer was
removed. The aqueous layer was extracted with CHCl3 (0.5 L × 2). The combined organic layers
were washed with brine, and dried over MgSO4. After filtration, the solvent was evaporated in
vacuo. The residue was recrystallized from EtOH/Et2O to afford 2-(4-(2-hydroxyethyl)piperazin-
1-yl)-N-(6-methyl-2,4-bis(methylthio)pyridin-3-yl)acetamide (48) (71.9 g, 97%) as colorless
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crystals. Mp 119-120℃; IR (KBr) cm-1: 3336, 1687, 1564, 1534, 1478 ; H-NMR (CDCl3) :
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