Structure–activity relationship studies of atpenin A5 analogs with chemical modification of the side chain moiety

Article abstract of DOI:10.1016/j.tetlet.2019.03.026

We designed and synthesized new atpenin A5 analogs for SAR study. Most of the analogs lacked one or several functional groups in the side chain of atpenin A5, and the stereoisomers proved to be weak nematode complex II inhibitors. However, we determined t

Full text of DOI:10.1016/j.tetlet.2019.03.026

Accepted Manuscript
Structure–activity relationship studies of atpenin A5 analogs with chemical
modification of the side chain moiety
Masaki Ohtawa, Keisuke Yano, Atsuyoshi Miyao, Tohru Hiura, Kouhei
Sugiyama, Shiho Arima, Kiyoshi Kita, Satoshi Omura, Tohru Nagamitsu
PII:
S0040-4039(19)30233-3
https://doi.org/10.1016/j.tetlet.2019.03.026
TETL 50667
DOI:
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
1 February 2019
6 March 2019
8 March 2019
Please cite this article as: Ohtawa, M., Yano, K., Miyao, A., Hiura, T., Sugiyama, K., Arima, S., Kita, K., Omura,
S., Nagamitsu, T., Structure–activity relationship studies of atpenin A5 analogs with chemical modification of the
side chain moiety, Tetrahedron Letters (2019), doi: https://doi.org/10.1016/j.tetlet.2019.03.026
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Structure–activity relationship studies of
atpenin A5 analogs with chemical
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modification of the side chain moiety.
Masaki Ohtawa, Keisuke Yano, Atsuyoshi Miyao, Tohru Hiura, Kouhei Sugiyama, Shiho Arima, Kiyoshi Kita,
Satoshi Omura, and Tohru Nagamitsu

1
Tetrahedron Letters
Structure–activity relationship studies of atpenin A5 analogs with chemical
modification of the side chain moiety.
a
a
a
a
a
a
Masaki Ohtawa , Keisuke Yano , Atsuyoshi Miyao , Tohru Hiura , Kouhei Sugiyama , Shiho Arima ,
b
c
a
Kiyoshi Kita , Satoshi Omura , and Tohru Nagamitsu 
a
Department of Synthetic Natural Products Chemistry and Medicinal Research Laboratories, School of Pharmacy, Kitasato University, 5–9–1 Shirokane,
Minato-ku, Tokyo 108–8641, Japan
b
School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki, 108-8641, Japan
c
Kitasato Institute for Life Sciences, Kitasato University, Tokyo, 108-8641, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We designed and synthesized new atpenin A5 analogs for SAR study. Most of the analogs
lacked one or several functional groups in the side chain of atpenin A5, and the stereoisomers
proved to be weak nematode complex II inhibitors. However, we determined that 4-epi-atpenin
A5 was a potent nematode complex II inhibitor comparable to atpenin A5. Therefore, 4-epi-
atpenin A5 is expected to become a new lead compound in nematicide development.
Available online
2
009 Elsevier Ltd. All rights reserved.
Keywords:
Structure–activity relationship study
Atpenin A5
4
-epi-Atpenin A5
Nematode complex II inhibitor
6
1
. Introduction
The atpenins A5 (1), A4 (2), and B (3), which include a highly
atpenin A5 (1) has also been achieved. It is clear that atpenins
and their analogs are useful chemical tools for the elucidation of
complex II functionality. Moreover, they could act as lead
compounds for the development of novel helminth complex II-
specific inhibitors. In 2009, we achieved a stereoselective total
functionalized pyridine unit, were first isolated in 1988 from a
fermentation broth of the atpenin-producing strain Penicillium sp.
FO-125. These were used as growth inhibitors of both fatty acid
synthase deficient (A-1) and acyl-CoA synthase I deficient (L-7)
mutants of Candida lipolytica, and 3 was shown to inhibit the
ATP-generating system of Raji cells. Harzianopyridone (4) was
originally isolated from Trichoderma harzianum in 1989,
showing antifungal, antibacterial, and herbicidal activities.
NBRI23477 B (5) was isolated in 2005 from a culture broth of
Penicillium atramentosum PF1420 and is used as a growth
inhibitor of human prostate cancer cells (Figure 1). The absolute
configurations of atpenins A5 (1) and A4 (2) have been
10
synthesis of atpenin A5 (1) by improving Quéguiner’s
procedure. Then, we reported the total synthesis of other natural
atpenin A5 analogs (A4 (2) and B (3)), harzianopyridone (4), and
NBRI23477 B (5) using this synthetic strategy; the absolute
structures of 3, 4, and 5 were also determined. Next, we focused
on the structure–activity relationship of atpenin A5 and embarked
on the synthesis of new atpenin A5 analogs with chemical
modification of the side chain moiety because an inhibitory
activity against nematode complex II of the synthesized atpenins
A4 (2) and B (3), lacked one or two chloro groups in the side
chain of 1, reduced greatly (Table 1). It was shown that presence
of the side chain is important for the nematode complex II
inhibitory activity of 1. Recently, new findings that differed from
our previous results were reported by the Carreira group, in
which a new total synthesis of 1 using novel methodology that
they developed was achieved and led to the synthesis of new
analogs possessing a complex II inhibitory activity similar to that
1
,2
11
3
,4
5
6
,7
confirmed by X-ray crystallographic analysis. The total
synthesis of (±)-atpenin B (3) was reported by the Quéguiner
group in 1994. In 2003, new interest developed in atpenins A5
8
(1) and A4 (2) and harzianopyridone (4) when the compounds
were reported to provide high levels of inhibition in a microbial
screening assay against mitochondrial complex II (succinate-
ubiquinone oxidoreductase), which is an attractive target for the
9
treatment of helminthiasis. In particular, atpenin A5 (1) proved
12
of atpenin A5. Herein, we report structure–activity relationship
studies of atpenin A5 analogs with chemical modification of the
side chain moiety.
to be much more potent against bovine heart complex II than any
known complex II inhibitors, although the inhibition of 1 was
non-selective across helminthes and mammals. Crystal structure
analysis of Escherichia coli complex II co-crystallized with
—

——
Corresponding author. Tel.: +81-3-5791-6376; fax: +81-3-3444-4742; e-mail: nagamitsut@pharm.kitasato-u.ac.jp

2
Figure 1 Structures of the natural atpenin family: atpenins A5 (1), A4 (2), and B (3); harzianopyridone (4); and NBRI23477B (5).
OMOM
HO
Cl
MeO
MeO
I
8
steps
0%
2
N
N
OMOM
OH
N
O
Cl
6
7
MeO
MeO
2
4
6
Cl
3
steps
6%
5
6
OH
O
Cl
Cl
1
4 steps
9%
Atpenin A5 (1)
OHC
HO
OMe
2
8
9
Scheme 1 Total synthesis of atpenin A5 (1), reported previously by our group.
Figure 2 Designed new atpenin A5 analogs 10–17.
stereoselectively in 85% yield. Protection of the hydroxy group
as a trityl ether (87% yield) followed by epoxide-opening
reaction with Me CuLi and BF ·Et O afforded 22 in 75% yield.
Deprotection of the trityl ether 22 (44% yield) and dichlorination
of the resulting diol (76% yield) gave dichloride 23. Treatment of
2
. Results and discussion
The synthetic strategy for our previous total synthesis of
2
3
2
1
0
atpenin A5 (1) is convergent and amenable to the synthesis of a
variety of atpenin A5 analogs. Here, we designed new atpenin A5
analogs 10–17 for SAR study (Figure 2). The analogs 10–14
lacked one or several functional groups in the side chain of 1, and
the analogs 15–17 were stereoisomers of 1. Initially, synthesis of
2
3 with TBAF furnished the corresponding alcohol in 80% yield,
which was oxidized with TEMPO to give aldehyde 24 in 77%
yield. The key coupling reaction of the pyridine unit 7 with the
aldehyde 24 followed by Dess–Martin oxidation gave the
corresponding ketone in 43% yield over two steps. Finally,
MOM deprotection under acidic conditions using TFA afforded
2
-demethylatpenin A5 (10) was investigated (Scheme 2). In our
10
previous total synthesis , the 2-methyl group and its
stereochemistry were derived from a commercially available
chiral Roche ester (8). Therefore, the starting material was
changed for the synthesis of 10. 1,4-Butanediol (18) was
protected as a mono TIPS ether and the resulting alcohol was
oxidized with IBX to give aldehyde 19 in 71% over two steps.
Wittig olefination of 19 afforded (E)-,-unsaturated ester 20
quantitatively. Other chemical transformations to afford the
desired 2-demethylatpenin A5 (10) followed our previous total
synthesis. DIBAL reduction of 20 gave the corresponding allyl
alcohol in 79% yield, which was subjected to Sharpless
asymmetric epoxidation using (–)-DET to give epoxy alcohol 21
2
-demethylatpenin A5 (10) quantitatively.

3
Scheme 3 Synthesis of 4-demethylatpenin A5 (11).
Scheme 2 Synthesis of 2-demethylatpenin A5 (10).
Next, 4-demethylatpenin A5 (11) was synthesized (Scheme 3).
1
0
In our previous total synthesis , the key intermediate 25 was
1
3
subjected to a reductive epoxide-opening reaction with LiEt BH
3
to give the alcohol quantitatively and regioselectively. After
deprotection of trityl ether (84% yield), dichlorination of the
resulting diol followed by TIPS deprotection afforded alcohol 28
in 62% yield over two steps. The alcohol 28 was converted to
aldehyde 29 in 74% yield by treatment with TEMPO. The
coupling reaction of the pyridine unit 7 and Dess–Martin
oxidation (65% yield over two steps) followed by MOM
deprotection under acidic conditions (quant.) afforded 4-
demethylatpenin A5 (11).
Scheme 4 Synthesis of 5-dechloroatpenin A5 (12).
The synthesis of 2,4-didemethylatpenin A5 (13) is shown in
As our next target, 5-dechloroatpenin A5 (12) was selected
Scheme 4). In our previous total synthesis , the key
intermediate 30 was converted to alcohol 31 in 33% yield over
three steps via Barton–McCombie deoxygenation and
deprotection of the trityl group. Chlorination of 31 (83% yield),
deprotection of TIPS ether (96% yield), and TEMPO oxidation
1
0
Scheme 5. The epoxide-opening reaction of a known epoxide 33
(
14
with a freshly prepared Grignard reagent 34 in the presence of
15
Li CuCl followed by deprotection of TBDPS ether with TBAF
2
4
afforded diol 35 quantitatively and regioselectively. Treatment of
5 with NCS and PPh gave dichloride 36 in 82% yield.
3
3
Subsequent acidic hydrolysis furnished the corresponding
aldehyde. The coupling reaction of the pyridine unit 7 with the
aldehyde followed by Dess–Martin oxidation afforded the
corresponding ketone in 62% yield over three steps. Final MOM
deprotection under acidic conditions gave 2,4-didemethylatpenin
A5 (13) in 71% yield.
(
69% yield) afforded aldehyde 32. The subsequent coupling
reaction of the pyridine unit 7 followed by Dess–Martin
oxidation afforded the corresponding ketone in 19% yield over
two steps. This was followed by MOM deprotection under acidic
conditions (88%) to afford 5-dechloroatpenin A5 (12).

4
Scheme 5 Synthesis of 2,4-didemethylatpenin A5 (13).
The synthesis of 2,4-didemethyl-5-dechloroatpenin A5 (14) is
shown in Scheme 6. The requisite aldehyde 39, a coupling
partner of 7, was synthesized from a commercially available diol
Scheme 6 Synthesis of 2,4-didemethyl-5-dechloroatpenin A5
(14).
Next, synthesis of atpenin A5 stereoisomers was investigated.
Synthesis of 2-epi-atpenin A5 (15) was achieved without any
difficulty in a manner similar to that used in our previous total
synthesis of 1, in which only the starting material was changed
from (–)-Roche ester (8) to the antipode 41 (Scheme 7).
3
8 via mono-TBDPS protection (96%) and TEMPO oxidation
(89%). The coupling reaction of the pyridine unit 7 and Dess–
Martin oxidation afforded the corresponding ketone in 64% yield
over two steps. Subsequent deprotection with TBAF gave
ketoalcohol 40 in 85% yield. Finally, chlorination and MOM
deprotection gave rise to 2,4-didemethyl-5-dechloroatpenin A5
(14) in 75% yield over two steps.
Scheme 7 Synthesis of 2-epi-atpenin A5 (15).

5
Scheme 9 Synthesis of 5-epi-atpenin A5 (17).
Table 1. Inhibitory activity of the atpenin A5 analogs against
a)
nematode complex II.
b)
IC50 (nM)
Atpenin A5 (1)
Atpenin A4 (2)
Atpenin B (3)
14
110
2100
315
1
1
1
1
1
0
1
2
3
4
Scheme 8 Synthesis of 4-epi-atpenin A5 (16).
201
Next, synthesis of 4-epi-atpenin A5 (16) was investigated
(
Scheme 8). The key intermediate 49 in our previous total
48.3
10
synthesis was subjected to Sharpless asymmetric epoxidation
using (+)-DET to give epoxide 50 in 85% yield. Protection of 50
as a trityl ether (quant.) and the epoxide-opening reaction (89%)
led to alcohol 51. To introduce the C5-chloro group with correct
stereochemistry, steric inversion of the C5-hydroxy group of 51
607
754
2000
7
15
1
6
was required. Therefore, the Mitsunobu reaction of 51 followed
by methanolysis of the resulting ester was conducted to give 52
in 71% over two steps. Birch reduction of 52 afforded diol 53 in
1
6
17
22.4
8
0% yield. Chlorination of 53 and deprotection of TIPS ether
a)
b)
The nematode complex II is derived from Ascaris suum. All
afforded alcohol 54 in 75% yield over two steps. The coupling
reaction of the pyridine unit 7 with aldehyde 55, which was
derived from 54 by TEMPO oxidation, followed by Dess–Martin
oxidation afforded the corresponding ketone in 15% yield over
three steps. Final deprotection with TBAF gave the 4-epi-atpenin
A5 (16) in 86% yield.
inhibitory activities against nematode complex II (IC50 values) are
the averages of three independent experiments.
The nematode complex II inhibitory activities of the new
analogs 10–14 (which lacked one or several functional groups in
the side chain of 1), together with the stereoisomers 15–17, are
shown in Table 1. Except for 12, the analogs 10–14
1
7
To synthesize 5-epi-atpenin A5 (17) (the final target for this
SAR), Mitsunobu reaction of the key intermediate 30 in our
previous total synthesis was conducted (Scheme 9). After
hydrolysis of the resulting ester, the desired alcohol 56 was
obtained in 80% yield over 2 steps. Deprotection of the trityl
ether (76%), dichlorination (92%), and deprotection of the TIPS
ether (69%) afforded alcohol 57. The coupling reaction of the
pyridine unit 7 with aldehyde 58, which was prepared by
TEMPO oxidation of 57, followed by Dess–Martin oxidation
furnished the corresponding ketone in 34% yield over three steps.
Deprotection with TBAF gave 5-epi-atpenin A5 (17) in 97%
yield.
significantly reduced the nematode complex II inhibitory activity.
This result indicates that each functional group (except for the 5’-
chloro group) in the side chain of 1 is important in chemical
interaction at the binding site with 1 in the nematode complex II.
This result is very different from that of SAR studies of 1 toward
1
8,19
mammalian complex II.
Furthermore, the nematode complex
II inhibitory activity of the 2-epi-atpenin A5 analog 15 also
decreased greatly, whereas those of the 5-dechloroatpenin A5
analog 12 and 5-epi-atpenin A5 analog 17 decreased only slightly.
Among the new atpenin A5 analogs synthesized here, the 4-epi-
atpenin A5 analog 16 showed the strongest nematode complex II
inhibitory activity, which was comparable to 1. Therefore, the
order of stereochemistry importance of 1 for nematode complex
II inhibitory activity was C2 >> C5 > C4; for C2, the
stereochemistry of 1 was shown to be particularly important.

6
Bioorganic & Medicinal Chemistry
4
5
6
.
.
.
Cutler, H. G.; Jacyno, J. M. Agric. Biol. Chem. 1991, 55, 2629–
631.
In conclusion, we designed and synthesized new atpenin A5
2
analogs 10–17 for SAR study. Most of the synthesized atpenin
A5 analogs reduced the nematode complex II inhibitory activity.
However, stereoisomer 16 was found to be a potent nematode
complex II inhibitor that was comparable to 1. Therefore, 4-epi-
atpenin A5 (16) is expected to be useful as a new lead compound
for the development of nematicides. We have begun to develop a
concise and practical synthesis of 4-epi-atpenin A5 (16) for large
scale synthesis. Additional structure–activity relationship studies
of 1 and a new synthesis of 16 are in progress and will be
reported in due course.
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1
1. Ohtawa, M.; Sugiyama, K.; Hiura, T.; Izawa, S.; Shiomi, K.;
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This work was supported by a Grant-in-Aid for Scientific
Research (C) (S.A., KAKENHI No. 17K08370), a Grant-in-Aid
for Young Scientists (B) (M.O., KAKENHI No. 21790117 and
12. Krautwald, S.; Nilewski, C.; Mori, M.; Shiomi, K.; Omura, S.;
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2
3790140), and a Kitasato University research grant for young
researchers (M.O.). We also thank Ms. N. Sato and Dr. K. Nagai
Kitasato University) for kindly measuring NMR and MS
1
4. Zheng, H.; Zhao, C.; Fang, B.; Jing, P.; Yang, J.; Xie, X.; She, X.
(
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7. Enzyme assays: The nematode complex II (NADH-fumarate
reductase, derived from Ascaris suum) activity was measured in
assay mixtures containing 50 mM potassium phosphate (pH 7.2),
10 mM -D-glucose, 20 units of glucose oxidase, 26 units of
catalase and 200 M NADH. The reaction is started by the
addition of sodium fumarate (5 mM). The absorbance change at
Appendix A. Supplementary Material
Supplementary data for this article can be found online at.
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Highlights
Structure–activity relationship studies of atpenin
A5 analogs
Synthesis of atpenin A5 analogs with chemical
modification of the side chain moiety
4
’-epi-Atpenin A5, a potent nematode complex II
inhibitor