Synthesis and structure-activity relationship of pyripyropene A derivatives as potent and selective acyl-CoA:cholesterol acyltransferase 2 (ACAT2) inhibitors: Part 3

Article abstract of DOI:10.1016/j.bmcl.2013.04.075

In an effort to develop potent and selective inhibitors toward ACAT2, structure-activity relationship studies were carried out using derivatives based on pyripyropene A (PPPA, 1). In particular, we investigated the possibility of introducing appropriate 1,11-O-benzylidene and 7-O-substituted benzoyl moieties into PPPA (1). The new o-substituted benzylidene derivatives showed higher selectivity for ACAT2 than PPPA (1). Among them, 1,11-O-o-methylbenzylidene-7-O- p-cyanobenzoyl PPPA derivative 7q and 1,11-O-o,o-dimethylbenzylidene-7-O-p- cyanobenzoyl PPPA derivative 7z proved to be potent ACAT2 inhibitors with unprecedented high isozyme selectivity.

Full text of DOI:10.1016/j.bmcl.2013.04.075

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3798–3801
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationship of pyripyropene
A derivatives as potent and selective acyl-CoA:cholesterol
acyltransferase 2 (ACAT2) inhibitors: Part 3
Masaki Ohtawa a, Hiroyuki Yamazaki a, Satoshi Ohte a, Daisuke Matsuda a, Taichi Ohshiro a,b
,
b
c
a,
⇑
⇑
Lawrence L. Rudel , Satoshi Omura , Hiroshi Tomoda , Tohru Nagamitsu a,
¯
a Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
b Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
c Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to develop potent and selective inhibitors toward ACAT2, structure–activity relationship
studies were carried out using derivatives based on pyripyropene A (PPPA, 1). In particular, we investi-
gated the possibility of introducing appropriate 1,11-O-benzylidene and 7-O-substituted benzoyl moie-
ties into PPPA (1). The new o-substituted benzylidene derivatives showed higher selectivity for ACAT2
than PPPA (1). Among them, 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl PPPA derivative 7q and
1,11-O-o,o-dimethylbenzylidene-7-O-p-cyanobenzoyl PPPA derivative 7z proved to be potent ACAT2
inhibitors with unprecedented high isozyme selectivity.
Received 13 April 2013
Revised 26 April 2013
Accepted 29 April 2013
Available online 8 May 2013
Keywords:
ACAT2
ACAT2-selective inhibitor
Pyripyropene A
Ó 2013 Elsevier Ltd. All rights reserved.
Antiatherosclerotic agent
Structure–activity relationship studies
Acyl-CoA:cholesterol acyltransferase (ACAT) plays an important
role in cholesterol metabolism in mammals. Recent molecular bio-
logical studies revealed the presence of two ACAT isozymes, ACAT1
and ACAT2, which have different functions in mammals.1–4 ACAT1
is ubiquitously expressed in tissues and cells such as sebaceous
glands, steroidogenic tissues, and macrophages, whereas ACAT2
is predominantly expressed in the liver and intestine.5 Conse-
quently, ACAT2-selective inhibitors could be employed as effective
cholesterol-lowering or anti-atherosclerotic agents, with fewer
side-effects than ACAT1-selective inhibitors. A newly developed
cell-based assay using ACAT1- or ACAT2-expressing CHO cells6,7
confirmed that pyripyropene A (PPPA, 1) is a potent and selective
inhibitor of ACAT2. Recent clinical studies showed that synthetic
avasimibe and pactimibe, which can inhibit both ACAT1 and
ACAT2, do not attenuate the progression of atherosclerosis.8,9 This
may be because the inhibition of ACAT1 in vascular cells, including
macrophages, causes the excessive accumulation of free choles-
terol in the cells and thus cytotoxicity.9 Very recently, 1 was pro-
ven to be orally active in an in vivo atherogenic mouse
model.10,11 Therefore, our group re-investigated the synthesis of
ACAT2-selective inhibitors based on PPPA derivatives for the devel-
opment of cholesterol-lowering or anti-atherosclerotic agents.
We have previously described structure–activity relationship
(SAR) studies of PPPA derivatives with a variety of substituted ben-
zoyl groups at the 7-position12 and 7-O-p-cyanobenzoyl PPPA
derivatives with various acyl groups at the 1- and 11-positions.13
As shown in Figure 1, 7-O-p-cyanobenzoyl PPPA derivative 2,
which exhibited higher ACAT2-innibitory activity (77 times) and
isozyme selectivity (4.6 times) than PPPA (1), was developed based
on results from our previous SAR studies. To the best of our knowl-
edge, 2 is the most potent ACAT2-inhibitor known, with higher iso-
zyme selectivity than 1.
For the current SAR study, we focused on new 1,11-O-benzyli-
dene-7-O-monosubstituted benzoyl PPPA derivatives. The ACAT2
inhibitory activity of 1,11-O-benzylidene acetal derivatives 3 and
4 synthesized during earlier SAR studies14,15 was comparable to
1, whereas the PPPA derivatives showed lower isozyme selectivity
than 1. Herein, we report SAR studies of 1,11-O-benzylidene-7-O-
monosubstituted benzoyl PPPA derivatives, as well as the discov-
ery of new PPPA derivatives with more potent ACAT2 inhibitory
activity than 1 and unprecedented high isozyme selectivity.
The 1,11-O-benzylidene-7-O-monosubstituted benzoyl PPPA
derivatives 7 were prepared from pyripyropene tetraol 5 in two
steps as shown in Scheme 1.14
⇑
Corresponding authors. Tel.: +81 357916241 (H. Tomoda), +81 357916376
(T. Nagamitsu).
E-mail addresses: tomodah@pharm.kitasato-u.ac.jp (H. Tomoda), nagamitsut@
pharm.kitasato-u.ac.jp (T. Nagamitsu).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.075

M. Ohtawa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3798–3801
3799
Table 1
ACAT1 and 2 inhibitory activity and isozyme selectivity of 1,11-O-benzylidene-7-O-
O
H
O
N
O
H
O
N
monosubstituted benzoyl PPPA derivatives 7a–p (R2 = Ph)
HO
H
HO
H
Compound
IC50 (lM)
O
O
O
O
No.
R3
ACAT1
ACAT2
SI*
466.7
O
O
O
O
1
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
PPPA (1)
p-CN
p-F
p-Cl
p-Br
m-F
p-OMe
m-Cl
m-CN
m-Br
p-CHO
o-F
p-NO2
m-OMe
—
2.80
6.20
5.80
6.90
3.00
6.90
2.70
2.20
4.30
5.50
2.70
13.90
4.30
>80
0.0060
0.0070
0.0090
0.0100
0.0130
0.0140
0.0190
0.0300
0.0300
0.0300
0.0500
0.0600
0.0700
0.0700
0.1100
0.3200
0.3200
O
O
7
885.7
644.4
690.0
230.8
492.9
142.1
73.3
143.3
183.3
54.0
11
O
O
CN
O
O
2
PPPA (1)
IC50 (μΜ)
IC50 (μΜ)
>80
4.16
ACAT1
ACAT2
ACAT1
ACAT2
0.07
0.0009
>1000
4622
SI*
SI*
231.7
61.4
>1000.0
16.2
3 (R1 = H)
IC50 (μΜ)
7n
7o
7p
o-Cl
o-CN
o-OMe
1.78
0.79
3.40
O
O
N
2.5
10.5
4.0
ACAT1
ACAT2
HO
0.02
200
SI*
The PPPA derivatives are sorted in descending order based on ACAT2 inhibitory
activity.
Selectivity Index (SI): IC50 (ACAT1)/IC50 (ACAT2).
O
O
O
H
H
*
4 (R1 = Me)
IC50 (μΜ)
O
3.2
ACAT1
ACAT2
O
0.01
R1
isozyme selectivity (SI = 885.7) comparable to that of 1. The results
indicate that 7-O-p-cyanobenzoyl and 7-O-p-halobenzoyl groups
are requisite for high ACAT2 inhibitory activity and isozyme selec-
tivity, and that the position of the substituent on the phenyl group
in the benzoyl group is also critical: the ACAT2 inhibitory activity
decreased in the order of para-, meta-, and ortho-substitution.
These trends are consistent with those observed in our previous
SAR study.12
The new 1,11-O-benzylidene-7-O-monosubstituted benzoyl
derivatives 7a–m exhibited higher ACAT2 inhibitory activity than
1, but their SI value was lower. Therefore, the phenyl group in
the benzylidene acetal was chemically modified and additional
SAR studies were conducted. These studies lead to the selection
of the p-cyanobenzoyl and p-fluorobenzoyl groups as the 7-O-
substituent.12
Table 2 shows the ACAT2 inhibitory activity and isozyme selec-
tivity of new PPPA derivatives 7q–aa, synthesized as described in
Scheme 1. Derivatives 7q–v, with a methyl group on the phenyl
moiety in the benzylidene acetal, showed higher ACAT2 inhibitory
activity than 1, although only 1,11-O-o-methylbenzylidene-7-O-p-
cyanobenzoyl PPPA derivative 7q16 showed higher isozyme selec-
tivity (SI >6161) than PPPA (1) and 2. We assumed that the
320
SI*
Figure 1. Structures of PPPA (1), 7-O-p-cyanobenzoyl PPPA derivative 2, and 1,11-
O-benzylidene-7-O-p-pentanoyl PPPA derivatives 3 and 4 (⁄Selectivity Index (SI):
IC50 (ACAT1)/(IC50 (ACAT2)).
The ACAT2 inhibitory activity (IC50 values) and isozyme selec-
tivity (SI values) of 1 and synthetic derivatives 7a–p are listed in
Table 1. Most 1,11-O-benzylidene-7-O-monosubstituted benzoyl
derivatives 7 showed higher ACAT2 inhibitory activity than 1, ex-
cept 7n–7p, which have an ortho-substituent on the phenyl group
in the benzoyl moiety. Among PPPA derivatives 7a–m, 7-O-p-cya-
nobenzoyl derivative 7a provided the best ACAT2 inhibitory activ-
ity (IC50 = 0.0060
lM). 7-O-p-Halobenzoyl derivatives 7b–d, and
especially 7-O-p-fluorobenzoyl derivatives 7b, also showed high
O
O
N
O
H
O
N
HO
H
HO
H
a
O
O
H
5
HO
HO
OH
O
OH
R2
Table 2
O
ACAT1 and
2 inhibitory activity and isozyme selectivity of 1,11-O-o-substituted
6
benzylidene-7-O-monosubstituted benzoyl PPPA derivatives 7q–aa
Compound
R2
IC50 (lM)
No.
R3
ACAT1
ACAT2
SI*
7q
7r
7s
7t
7u
7v
7w
7x
7y
o-MePh
o-MePh
m-MePh
m-MePh
p-MePh
p-MePh
o-MeOPh
o-FPh
o,p-diMePh
o,o-diMePh
a-Naphthyl
p-CN
p-F
p-CN
p-F
p-CN
p-F
p-CN
p-CN
p-CN
p-CN
p-CN
—
>72.8
13.9
3.73
9.51
8.11
5.54
>71.0
0.33
10.0
>71.2
>69.1
>80
0.0118
0.0230
0.0078
0.0476
0.0097
0.0151
0.0368
0.0068
0.0066
0.0072
0.0232
0.0700
>6161
604.3
O
H
O
N
HO
478.2
199.8
836.1
366.9
>1929
48.5
1515
>9916
>2978
>1000.0
b
O
O
O
1
O
R3
H
11
R2
O
7
7z
7aa
PPPA (1)
Scheme 1. Reagents and conditions: (a) corresponding aldehydes or dimethylace-
tals, PPTS, DMF, rt; (b) corresponding aromatic carboxylic acids, EDCI, cat. DMAP,
CH2Cl2, rt, 7a–aa: 20–67%.
—
*
Selectivity Index (SI): IC50 (ACAT1)/IC50 (ACAT2).

3800
M. Ohtawa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3798–3801
Essential
R'
Essential
O
O
N
For ACAT2 inhibitory activity
Essential
diacetyl ≥
HO
o,o-dimethylbenzylidene acetal ≥
o-methylbenzylidene acetal ≥
benzylidene acetal
13
R
O
O
O
p-cyanophenyl ≥
p-halophenyl ≥
>> others
H
1
7
R'O
R
p-methoxyphenyl >
i-pentyl > phenyl = n-pentyl
> methyl >> others
For isozyme selectivity
o,o-dimethylbenzylidene acetal ≥
o-methylbenzylidene acetal ≥
benzylidene acetal ≥
diacetyl
H
11
R'O
>> others
Figure 2. Summary of our earlier and our latest SAR studies on synthetic PPPA derivatives.
presence of the o-substituent on the phenyl group in the benzyli-
dene acetal plays an important role in high isozyme selectivity
and thus synthesized other 1,11-O-o-substituted benzylidene-7-
O-p-cyanobenzoyl PPPA derivatives. Unfortunately, all attempts
to synthesize derivatives with substituents other than methyl,
methoxy or fluoro groups failed due to steric hindrance at the ortho
position. Although derivative 7x, with an o-fluoro group, showed
low isozyme selectivity, derivative 7w, with an o-methoxy group,
exhibited higher isozyme selectivity than 1, as expected. In
addition, we designed and synthesized new derivatives 7y (with
o,p-dimethyl groups) and 7z (with o,o-dimethyl groups). Both
derivatives exhibited better ACAT2 inhibitory activity and isozyme
selectivity than 1. In particular, o,o-dimethylbenzylidene deriva-
tive 7z proved to be a potent ACAT2 inhibitor with the highest iso-
Biomedical Innovation (NIBIO) (H.T.), and by a grant-in-aid for Sci-
entific Research (B) 18390008 (H.T.). The authors thank Meiji Seika
Pharma Co., Ltd. for their extensive cooperation, ChemGenesis
and PharmaDesign, Inc. for their valuable advice, and Chemical
Analysis Center (Kitasato Univ., Ms. Sato and Dr. Nagai) for kindly
obtaining NMR and MS spectra. The authors also thank Professor
Takahashi (Tokyo Institute of Technology) and Professor Doi
(Tohoku Univ.) for their helpful suggestions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.
075.
zyme selectivity (SI >9916) observed to date. The
a-naphthyl
derivative 7aa also showed higher isozyme selectivity than 1.
These results show that the o-substituent on the phenyl group in
the benzylidene acetal, and especially a methyl group, contributes
significantly to high isozyme selectivity.
Figure 2 summarizes all our ACAT2 inhibitory activity and iso-
zyme selectivity SAR results on synthetic PPPA derivatives. Our ini-
References and notes
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a-pyrone, and
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benzylidene acetal at the 1,11-dihydroxy position, augment ACAT2
inhibitory activity, but decrease isozyme selectivity. These groups
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our latest SAR study revealed that the p-cyanobenzoyl group at the
7-hydroxy position and o-substituted benzylidene acetal at the
1,11-dihydroxy position are superior to other substituents, and
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In conclusion, novel 1,11-O-benzylidene-7-O-monosubstituted
benzoyl PPPA derivatives were prepared and evaluated in cell-
based assays to measure ACAT1 and ACAT2 inhibition.17 Most of
the synthetic PPPA derivatives showed more potent ACAT2 inhib-
itory activity than natural PPPA (1). Among them, four derivatives
(7q, 7w, 7z, and 7aa) with an o-substituent on the phenyl group
in the benzylidene acetal exhibited higher isozyme selectivity
than 1. In particular, 1,11-O-o-methylbenzylidene-7-O-p-cya-
nobenzoyl PPPA derivative 7q and 1,11-O-o,o-dimethylbenzylid-
ene-7-O-p-cyanobenzoyl PPPA derivative 7z proved to be potent
ACAT2 inhibitors with unprecedented high isozyme selectivity
(SI: >6161 and >9916, respectively). The in vivo antiatherosclerot-
ic activity of these derivatives will be reported elsewhere. Further
SAR studies of the PPPA analogues are currently underway in our
laboratory.
9. Nissen, S. E.; Tuzcu, E. M.; Brewer, H. B.; Sipahi, I.; Nicholls, S. J.; Ganz, P.;
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12. Ohtawa, M.; Yamazaki, H.; Ohte, S.; Matsuda, D.; Ohshiro, T.; Rudel, L. L.;
¯
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¯
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Tomoda, H.; Nagamitsu, T. Bioorg. Med. Chem. Lett. 2013, 23, 2659.
¯
14. Obata, R.; Sunazuka, T.; Kato, Y.; Tomoda, H.; Harigaya, Y.; Omura, S. J. Antibiot.
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¯
Harigaya, Y.; Rudel, L. L.; Omura, S.; Tomoda, H. J. Antibiot. 2008, 61, 503.
16. Selected spectra data for 7q: ½a D24
ꢀ
+ 208 (c 1.0, CHCl3); IR (KBr) 3434, 2947, 2863,
2233, 1708, 1643, 1579, 1414, 1273, 1219, 933, 760 cmꢁ1
;
1H NMR (CDCl3,
400 MHz) d 8.97 (dd, 1H, H-200, J = 0.8, 3.2 Hz), 8.67 (dd, 1H, H-600, J = 2.0,
6.4 Hz), 8.22 (d, 2H, H–Ar, J = 11.2 Hz), 8.09–8.05 (m, 1H, H-400), 7.81 (d, 2H, H–
Ar, J = 11.2 Hz), 7.65–7.62 (m, 1H, H–Ar), 7.40–7.36 (m, 1H, H-500), 7.24–7.23
(m, 2H, H–Ar), 7.22–7.13 (m, 1H, H–Ar), 6.40 (s, 1H, H-50), 5.70 (s, 1H, CHAr),
5.30 (dd, 1H, H-7, J = 8.0, 12.8 Hz), 5.05 (dd, 1H, H-13, J = 3.2, 4.8 Hz), 3.90 (d,
1H, H-11a, J = 13.6 Hz), 3.58–3.52 (m, 2H, H-11b, J = 13.6 Hz), 3.00 (br d, 1H,
OH-13, J = 2.8 Hz), 2.40 (s, 3H, ArMe), 2.29 (d, 1H, H-3a, J = 17.2 Hz), 2.05–1.18
Acknowledgments
This work was supported by the Program for Promotion of Fun-
damental Studies in Health Sciences of the National Institute of

M. Ohtawa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3798–3801
3801
(m, 7H, H-2, 3b, 5, 8, 9), 1.86 (s, 3H, Me), 1.53 (s, 3H, Me), 1.26 (s, 3H, Me); 13
NMR (CDCl3, 100 MHz) 164.06, 163.86, 162.00, 157.45, 151.62, 146.78,
135.97, 135.53, 133.85, 132.93, 132.39, 130.42, 130.26, 128.86, 127.03, 126.01,
125.95, 123.63, 117.85, 116.77, 103.03, 100.76, 99.24, 85.81, 83.34, 79.42,
78.29, 60.39, 60.01, 54.67, 48.83, 38.38, 37.07, 36.50, 25.04, 23.14, 21.06, 18.77,
C
18.21, 16.72, 13.39.
d
ESI-LRMS m/z 689 (MH+); ESI-HRMS (TFA-Na) calcd for C41H41N2O8 689.2863
(MH+), found 689.2885 (MH+).
17. Tomoda, H.; Nagamitsu, T.; Matsuda, D.; Ohshiro, T.; Ohtawa, M. Omura, S. PCT
¯
Int. Appl. WO 2011122468, 2011.