Synthesis of neuroprotective cyclopentenone prostaglandin analogs: Suppression of manganese-induced apoptosis of PC12 cells

Article abstract of DOI:10.1016/j.bmcl.2006.12.004

The synthesis and evaluation for anti- and proapoptotic properties of cyclopentenone prostaglandin analogs are described. Novel J-type analogs of NEPP11 with a cross-conjugated cyclopentadienone moiety and a lipophilic ω-side chain suppressed manganese io

Full text of DOI:10.1016/j.bmcl.2006.12.004

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5487–5491
Synthesis of neuroprotective cyclopentenone prostaglandin analogs:
Suppression of manganese-induced apoptosis of PC12 cells
Kyoji Furuta,a Masahide Maeda,a Yoko Hirata,b Shoko Shibata,b
Kazutoshi Kiuchib and Masaaki Suzukia,c,
*
aRegeneration and Advanced Medical Science, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan
bDepartment of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
cMolecular Imaging Research Program, Frontier Research System, RIKEN, 6-7-3 Minatojima-minamimachi, Chuo-ku,
Kobe 650-0047, Japan
Received 27 October 2006; revised 25 November 2006; accepted 4 December 2006
Available online 15 December 2006
Abstract—The synthesis and evaluation for anti- and proapoptotic properties of cyclopentenone prostaglandin analogs are
described. Novel J-type analogs of NEPP11 with a cross-conjugated cyclopentadienone moiety and a lipophilic x-side chain
suppressed manganese ion-induced apoptosis of PC12 cells at comparable levels to NEPP11, while monoenone derivatives were
inactive. The proapoptotic activities of J-type analogs were much lower than that of NEPP11. Natural 15-deoxy-D12,14-PGJ2 and
D7-PGA1 methyl ester were highly toxic, inducing apoptosis at lower concentrations.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Prostaglandins (PGs) E and D are important lipid-
signaling mediators that are metabolized to cyclopen-
tenone PGs of the A and J series by both enzymatic
and non-enzymatic processes.1 Although cyclopente-
none PGs have no membrane receptors, they pene-
trate cell membranes and bind directly to
intracellular proteins, and thus regulate their activi-
ties. Cyclopentenone PGs are now attracting increas-
ing interest as low-molecular-weight compounds that
can modulate cellular proliferation, differentiation,
and viability.2,3
1 (designated NEPP11).6 EPP11 was found to protect
the brain in vivo against ischemic death caused by per-
manent middle cerebral artery occlusion in mice.5 In
contrast, natural 15-deoxy-D12,14-PGJ2 (2), which pos-
sesses an extended trienone moiety, could not protect
neuronal cells against glutamate toxicity.5
O
COOCH3
COOH
O
2, 15-deoxy-Δ12,14-PGJ2
1, NEPP11
In previous studies on the synthesis and biological activ-
ities of cyclopentenone PGs, we demonstrated that cer-
tain synthetic analogs with a cross-conjugated dienone
structure exhibited neuroprotective activities.4–7 These
dienone PGs not only protected HT22 neuronal cells
against oxidative glutamate toxicity, but also promoted
neurite outgrowth from PC12 cells or from dorsal root
ganglion explants induced by nerve growth factor. The
most potent compound for both activities was analog
More recently, we found that low concentrations of
NEPP11 <10 lM) inhibited manganese ion-induced
apoptosis of PC12 cells,8 which has been used as a cellu-
lar model of Parkinson’s disease.9 These findings sug-
gested that NEPP11 may act as a therapeutic agent
against neurodegenerative diseases including Alzhei-
mer’s and Parkinson’s diseases. At high concentrations
P10 lM), however, NEPP11 was toxic to PC12 cells,
as well as inducing apoptosis,8 suggesting the need for
less toxic compounds with sufficient antiapoptotic activ-
ity. The rational design of better neuroprotective com-
pounds thus requires investigation of the relationships
Keywords: Neuroprotective; Cyclopentenone prostaglandin; Apopto-
sis; Manganese ion; Perkinson’s disease.
*
Corresponding
suzukims@biomol.gifu-u.ac.jp
author.
Tel./fax:
+81
230
6502; e-mail:
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.004

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K. Furuta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5487–5491
between the structure of NEPP11 analogs and their anti-
and proapoptotic effects. This paper describes the syn-
thesis of modified NEPP11 analogs and evaluation of
their effects on manganese ion-treated/untreated PC12
cells.
Two different types of monoenone analogs, an alkyliden
cyclopentanone type compound 3 and an endo cyclo-
pentenone type compound 4, can be seen in the struc-
tures. Compounds 5, 6, and 7 are x-side-chain
variants, in which 5 has a simple octyl chain, 6 has the
same x-side chain as natural primary prostaglandins,
and 7 does not have an x-side chain. The remaining
compounds in Figure 1 represent the topological iso-
mers of A-type PGs at the cyclopentenone ring and
are categorized as J-type PGs. Thus, 8 and 9 are the up-
side-down isomers of NEPP11 and 5, respectively. We
also designed 10 and its enantiomer 11, each of which
contains a bromophenyl group at the terminal end of
the x-side chain. These brominated compounds are
intended to apply to positron emission tomography
studies as 76Br-incorporated tracers.
NEPP11 can protect neuronal cells from manganese ion-
induced apoptosis as well as glutamate-mediated cell
death (necrosis).5–7 We recently reported that induction
of hemeoxygenase-1 (HO-1) through activation of the
Keap1/Nrf2 pathway by NEPP11 is crucial for suppress-
ing glutamate-mediated necrotic death of HT22 cells.7
HO-1 is a phase II enzyme that serves as a defense sys-
tem against oxidative stress. In contrast, the antiapopto-
tic effect of NEPP11 on manganese ion-treated PC12
cells is associated with prevention of phosphorylation
of c-Jun N-terminal kinase (JNK).8 Since MKK4, an
upstream kinase of JNK, is activated by manganese
ion in the presence of NEPP11, one target of NEPP11
may be the phosphorylation stage of JNK by activated
MKK4. Taken together, these data suggest that
NEPP11 targets different molecules in each of the neuro-
protective effects. This implies that the structural fea-
tures of compounds required for the suppression of
manganese-induced apoptosis may differ from those re-
quired for protection against glutamate toxicity.
The designed cyclopentenone PG analogs were generally
synthesized based on the established three-component
coupling method.11 We have already described the syn-
theses of NEPP11, 5, and 6.6,12 The monoenone deriva-
tives 3 and 4 were prepared from NEPP11 and 13 as
illustrated in Scheme 1. Thus, conjugate reduction of
NEPP11 with DIBAL–CuI–CH3Li complex13 proceed-
ed selectively at C10–C11 to yield the alkyliden-type
analog 3. In contrast, treatment of 13 with tributylstann-
ane in the presence of di-tert-butylperoxide gave the sat-
urated derivative 14,14 which was then converted to the
desired product 4 by acidic elimination of the oxygen
functional group at C11. The remaining A-type analog
7 was synthesized by aldol condensation of cyclopent-
2-en-1-one with 7-oxoheptanoate followed by the mesy-
lation-elimination sequence.
We reported previously that two structural aspects of
cyclopentenone PGs are essential for the suppression
of glutamate-induced neuronal cell death: a cross-conju-
gated dienone moiety and a modestly lipophilic x-side
chain.5 To explore simple relationships between the
structure and antiapoptotic effects of cyclopentenone
PGs on manganese ion-treated PC12 cells, we designed
and synthesized several NEPP11 analogs, particularly
focusing on the enone and x-side-chain moieties.10 The
structures of the compounds investigated are presented
in Figure 1.
The J-type derivatives were synthesized by employing a
modified three-component coupling method, in which
the roles of the side-chain units were reversed. This pro-
cess involves nucleophilic attack of the organocuprate or
organozincate derived from the a-side-chain unit and
subsequent trapping of the resulting enolate with the
x-side-chain aldehyde. Scheme 2 demonstrates the syn-
thesis of 8 by this method. The orthoester 16, selected
as the a-side-chain unit, was prepared as described.15
The x-side-chain aldehyde 17 was synthesized by (1)
Friedel–Crafts acylation of toluene with 6-bromohexa-
noyl chloride, followed by reductive deoxygenation of
the resulting ketone with triethylsilane in one pot, (2)
conversion of the bromide, 6-(4-methylphenyl)-1-
bromohexane, to the corresponding iodide with sodium
iodide, and (3) DMSO oxidation of the iodide to the
desired aldehyde 17. The three-component coupling of
the cuprate generated from 16, (R)-4-(tert-butyldimeth-
ylsiloxy)cyclopent-2-en-1-one, and 17 afforded the aldol
adduct 18 in the ester form through concomitant
hydrolysis of the orthoester moiety during workup pro-
cedures. The adduct 18 was dehydrated with metha-
nesulfonyl chloride and 4-(dimethylamino)pyridine,
followed by elimination of the siloxy moiety under acid-
ic conditions to produce 8. The use of octanal or 5-(4-
bromophenoxy)pentanal as the x-side-chain unit yielded
the derivatives 916 (38% in three steps) and 10 (26% in
three steps), respectively. Compound 11, the antipode
of 10, was similarly synthesized using the enantiomeric
O
O
COOCH3
COOCH3
COOCH3
COOCH3
4
6
3
O
O
O
OH
5
7
COOCH3
COOCH3
COOCH3
O
O
O
O
9
8
COOCH3
Br
COOCH3
Br
O
O
11
10
Figure 1. Structures of cyclopentenone PGs.

K. Furuta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5487–5491
OH
5489
OHC
COOCH3
O
O
O
COOCH3
COOCH3
a
LnM
OTBDMS
OTBDMS
d
TBDMSO
12
13
O
O
COOCH3
COOCH3
b
b
c
4
3
OTBDMS
1
14
OH
O
O
COOCH3
e
a
7
15
Scheme 1. Synthesis of A-type PG analogs. Reagents and conditions: (a) MsCl, 4-(dimethylamino)pyridine, CH2Cl2, rt, 79% for 13 (16 h) and 87%
for 7 (2 h); (b) CH3COOH–THF–H2O, 70 ꢁC, 73% for 1 (22 h) and 87% for 4 (24 h); (c) CuI, CH3Li, DIBAL, THF–HMPA, À50 ꢁC, 1 h, 92%; (d)
Bu3SnH, (CH3)3COOC(CH3)3, 110 ꢁC, 15 m, 74%; (e) LTMP, THF, À78 ꢁC, 45 m, then methyl 7-oxoheptanoate, À78 ꢁC, 1.75 h, 58%.
O
Br
a
b, c
OHC
C(OCH3)3
d
Br
I
Cl
16
17
C(OCH3)3
LnM
TBDMSO
O
TBDMSO
O
COOCH3
e
f, g
8
OHC
OH
17
18
Scheme 2. Synthesis of J-type PG analogs. Reagents and conditions: (a) AlCl3, toluene, CH2Cl2, rt, 14 h, then Et3SiH, rt, 40 m, 93%; (b) NaI,
acetone, rt, 14 h, 94%; (c) DMSO, NaHCO3, 150 ꢁC, 10 m, 73%; (d) t-BuLi, CuI, PBu3; (e) ether–THF, À78 ꢁC (0.5 h) to À40 ꢁC (0.5 h), 30%; (f)
MsCl, 4-(dimethylamino)pyridine, CH2Cl2, rt, 24 h, 56%; (g) CH3COOH–THF–H2O, 70 ꢁC, 12 h, 72%.
cyclopentenone unit with the 4S configuration (32% in
three steps). All compounds synthesized were fully
Mn2+(–) Mn2+(+) Mn2+(–) Mn2+(+) Mn2+(–) Mn2+(+)
characterized by 400 MHz 1H NMR and 100 MHz
13C NMR.
A
B
C
The ability of the cyclopentenone PGs to inhibit manga-
nese ion-induced apoptosis of PC12 cells was evaluated
by DNA fragmentation.8 Briefly, PC12 cells (ca. 2·107
were preincubated with each PG (10 and 20 lM) at
37 ꢁC for 30 min in Dulbecco’s modified Eagle’s medium
supplemented with 5% fetal bovine serum, followed by
the addition of 0.25 mM MnCl2 and further incubation
for 20 h at the same temperature. The soluble DNA
was extracted from the cells, electrophoresed on agarose
gels, and visualized with an ultraviolet transilluminator.
Proapoptotic activity of the PGs was evaluated under the
same assay conditions, but in the absence of manganese
ion. The results are summarized in Figure 2 and Table 1.
0
10 20 0 10 0 10 20 0 10 0 10 20 0 10 20
Figure 2. DNA fragmentation patterns observed with compounds 2
(A), 5 (B), and 9 (C). PC12 cells were preincubated with each
compound (0, 10, and 20 lM) for 30 min and incubated with medium
in the presence (Mn2+(+)) or absence (Mn2+(À)) of MnCl2. After 20 h,
DNA fragmentation was measured.
ing that the cross-conjugated cyclopentadienone
structure is a prerequisite for exerting antiapoptotic ef-
fects. However, possession of the cross-conjugated die-
none structure alone is not sufficient, inasmuch as
D7-PGA1 methyl ester (6) and 7, both of which possess
the dienone moiety, showed no antiapoptotic activity.
These results indicate that the lipophilic interaction of
the x-side chain with the target protein is important
for binding. Thus, the hydroxy group in the x-side chain
Compound 5 suppressed manganese-induced DNA
fragmentation to a similar extent as NEPP11. At
10 lM, compound 5 was less toxic than NEPP11,
whereas at 20 lM, compound 5 induced a considerable
degree of DNA fragmentation (Fig. 2B). The monoe-
none analogs, 3 and 4, were completely inactive, indicat-

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K. Furuta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5487–5491
Table 1. Anti- and proapoptotic activities of cyclopentenone PGs
PGs. This covalent adduct formation may impair pro-
tein function, thus affecting cellular viability. Actually,
15-deoxy-D12,14-PGJ2 has been reported to target mito-
chondria respiratory complex I by covalent binding,
inhibiting the function of the protein and resulting in
the induction of reactive oxygen species.18 Although
the molecular mechanisms underlying the proapoptotic
effects of cyclopentenone PGs on PC12 cells are unclear,
differences in toxicity may partially be attributable to
the reactivity of the enone structures. This hypothesis
is supported by our finding, that compounds 3 and 4,
which have a less reactive monoenone structure, did
not exhibit cytotoxicity at the concentrations tested.
However, compound 6 was more toxic than NEPP11
and 5, suggesting that not only the cyclopentenone moi-
ety but also the structure of the x-side chain affected the
proapoptotic effects of these compounds. Conversely,
these findings suggest that appropriate modification of
the x-side chain and cyclopentenone moieties may elim-
inate the undesirable cytotoxicity of cyclopentenone
PGs. To achieve the desired functionality, it is necessary
to identify the target molecule(s) associated with the
proapoptotic effects and to establish structure–activity
relationships by rational structural modification. Analy-
sis of intracellular binding proteins using photoaffinity
labeling probes will be addressed in our future studies.
Compound
Antiapoa
Toxicityb
1
2
++
À
++
++
À
3
À
4
À
À
5
++
À
+
6
++
À
7
À
8
++
++
+
À
9
À
10
11
À
À
++
a Antiapoptotic activities: ca. 50% suppression of DNA fragmentation
at 10 lM (++); less than 50% (+); no suppression up to 20 lM (À).
b Proapoptotic activities: induction of extensive DNA fragmentation at
10 lM (++); slight fragmentation at 10 lM (+); no fragmentation up
to 20 lM (À).
may interfere with its interaction with the target protein,
and deletion of the x-side chain results in a complete
loss of binding affinity. In addition, since the octyl deriv-
ative 5 retains antiapoptotic activity, the terminal tolyl
group in the x-side chain is probably not an essential
component, but it may also not impair binding.
Natural 15-deoxy-D12,14-PGJ2 (2), which seems to meet
the above requirements except for differences in topolo-
gy and conjugation of the cyclopentenone structure,
strongly induced DNA fragmentation by itself, even at
low concentrations; thus, any antiapoptotic effect on
manganese ion-treated cells was not detected across
the range of concentrations examined (Fig. 2A). By con-
trast, compound 9, which possesses a conjugation sys-
tem saturated at the distal double bond of the
extended trienone structure of 2, showed a concentra-
tion-dependent inhibition of DNA laddering, but was
not toxic, even at 20 lM (Fig. 2C). The NEPP11-type
analog 8 also displayed similar activities. These results
indicate that the topological feature of the cyclopenta-
dienone structure is not important, at least for inhibition
of DNA fragmentation, whereas the extension of conju-
gation leads to enhanced toxicity. The bromophenoxy
analog 10 only weakly inhibited manganese-induced
apoptosis, whereas its enantiomer 11, which did not sup-
press DNA fragmentation, strongly induced apoptosis
at 10 lM, suggesting the inadequacy of the bulky or
excessively lipophilic bromophenoxy group. These find-
ings emphasize the regulatory role of the x-side chain in
binding.
In summary, we have assayed the simple structure–ac-
tivity relationship of cyclopentenone analogs. These
experiments have provided a greater understanding of
the importance of the x-side chain for both anti- and
proapoptotic effects. Moreover, we have succeeded in
elaborating novel J-type analogs with reduced cytotoxic-
ity. These compounds may not only be powerful molec-
ular tools to elucidate the mechanisms of Parkinson’s
disease but may also provide important clues to the
development of drugs for the treatment of such neurode-
generative diseases.
Acknowledgments
This work was supported in part by Grants-in Aid for
Scientific Research (B) (No. 17310124), Creative Scien-
tific Research (No. 13NP0401), and a consignment ex-
pense for Molecular Imaging Program on ‘Research
Base for Exploring New Drugs’ from the Ministry of
Education, Culture, Sports, Science and Technology
(MEXT), Japan.
References and notes
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react with nucleophiles such as thiols to form 1,4-ad-
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to thiols than are simple cyclopentenones.17 Therefore,
cyclopentenone PGs with a cross-conjugated dienone
moiety may react with a sulfhydryl group of a cysteine
residue in various proteins in the cells, forming covalent
complexes at lower concentrations than monoenone
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´
´
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