Discovery of novel (1S)-(-)-verbenone derivatives with anti-oxidant and anti-ischemic effects

Article abstract of DOI:10.1016/j.bmcl.2013.07.038

A series of novel (1S)-(-)-verbenone derivatives was synthesized bearing a 4-styryl scaffold. The synthesized compounds were tested for their anti-oxidant, anti-excitotoxic, and anti-ischemic activities. These derivatives significantly reduced oxygen-glucose deprivation-induced neuronal injury and N-methyl-d-aspartic acid-evoked excitotoxicity in cortical neurons. Furthermore, compound 3f was identified as a potent anti-ischemic agent in an in vitro ischemic model, potentially due to the inhibition of N-methyl-d-aspartic acid-evoked excitotoxicity and oxidative/nitrosative stress.

Full text of DOI:10.1016/j.bmcl.2013.07.038

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5421–5425
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel (1S)-(ꢀ)-verbenone derivatives with anti-oxidant
and anti-ischemic effects
Chung Ju a, , Sumi Song b, , Sunyoung Hwang a, Chorong Kim b, Minkyoung Kim b, Jail Gu b, Yu-Kyoung Oh c,
Kyeong Lee d, Jinsun Kwon b, Kiho Lee e, Won-Ki Kim a, , Yongseok Choi b,
⇑
⇑
a Department of Neuroscience, College of Medicine, Korea University, Anamdong-5-ga, Seongbuk-gu, Seoul 136-705, Republic of Korea
b Department of Biotechnology, School of Life Sciences and Biotechnology, Korea University, Anamdong-5-ga, Seongbuk-gu, Seoul 136-701, Republic of Korea
c College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
d College of Pharmacy, Dongguk University-Seoul, Seoul 100-715, Republic of Korea
e College of Pharmacy, Korea University, Sejong City 339-700, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel (1S)-(ꢀ)-verbenone derivatives was synthesized bearing a 4-styryl scaffold. The synthe-
Received 6 April 2013
Revised 18 June 2013
Accepted 17 July 2013
Available online 25 July 2013
sized compounds were tested for their anti-oxidant, anti-excitotoxic, and anti-ischemic activities. These
derivatives significantly reduced oxygen–glucose deprivation-induced neuronal injury and N-methyl-D-
aspartic acid-evoked excitotoxicity in cortical neurons. Furthermore, compound 3f was identified as a
potent anti-ischemic agent in an in vitro ischemic model, potentially due to the inhibition of
N-methyl-D-aspartic acid-evoked excitotoxicity and oxidative/nitrosative stress.
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Cerebral ischemia
Anti-oxidant activity
Anti-excitotoxic activity
Anti-ischemic activity
Verbenone
Stroke is one of the leading causes of death and long-term dis-
ability worldwide. Despite its high mortality rate and social-eco-
nomic burden, there are currently few treatment options
available for acute stroke. Ischemic stroke, which accounts for
80% of all strokes, is caused by a sudden interruption in cerebral
blood supply due to an embolus or a thrombus.1 Rapid restoration
of cerebral blood flow is critical to limit neuronal injury and conse-
quent loss of brain function. Thus, therapeutic intervention for
ischemic stroke generally entails prompt application of thrombo-
lytic agents such as tissue plasminogen activator (tPA). Although
tPA is the only thrombolytic agent approved by health authorities
thus far, it has clinical limitations, such as a narrow therapeutic
time window and a high risk of side effects.2 Recent evidence indi-
cates that neuronal injury in ischemic regions may progress even
after reperfusion.3 Therefore, the development of effective treat-
ments against this progressive ischemic injury has been eagerly
pursued to ensure high clinical efficacy.
glucose.3 Intracellular acidification and mitochondrial electron
leak induce the massive generation of reactive oxygen species
(ROS).4 The loss of ionic gradients and plasma membrane depolar-
ization trigger intracellular accumulation of glutamate, resulting in
excitotoxicity through over-activation of N-methyl-D-aspartate
(NMDA) receptors. Subsequently, NMDA receptor-mediated cal-
cium overload induces activation of neuronal nitric oxide synthase
(nNOS) and other proteases, further increasing oxidative/nitrosative
stress. Reperfusion is also reported to greatly increase production
of free radicals. In addition, activated microglia and infiltrating
peripheral leukocytes/monocytes produce ROS/reactive nitrogen
species (RNS), exacerbating inflammatory responses and ischemic
injury.4 Neurons are especially susceptible to ischemic injury,
expressing a higher density of glutamate receptors and possessing
relatively limited defense mechanisms (e.g., low ATP/ionic buffer-
ing and anti-oxidant capacity).5 Accordingly, an anti-ischemic
agent alleviating both excitotoxicity and ROS/RNS-associated in-
jury would possess considerable potential for synergistic benefits
for ischemia.6 Furthermore, the agents developed for this strategy
may also have alternative therapeutic perspectives when
employed in combination with a reperfusion remedy; either by
promoting penumbral survival until later initiation of reperfusion
therapy or by reducing secondary injury induced by reperfusion
following treatment with thrombolytic agents.7
The mechanisms underlying ischemic injury are diverse and
may occur sequentially and even simultaneously.3 Ischemic injury
is initiated by energy failure due to deprivation of oxygen and
⇑
Corresponding authors.
E-mail addresses: wonki@korea.ac.kr (W.-K. Kim), ychoi@korea.ac.kr (Y. Choi).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.038

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C. Ju et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5421–5425
(1S)-(ꢀ)-Verbenone is a naturally occurring anti-aggregation
Anti-oxidant capacities of verbenone derivatives were deter-
mined using two different types of chemical reactions: 2,2-diphe-
nyl-1-picrylhydrazyl (DPPH) assay and oxygen radical absorbance
capacity (ORAC) assay (Table 1) with vitamin C and trolox as stan-
dards, respectively.
pheromone generated by bark beetles from a host tree resin pre-
cursor,
a
-pinene.8 Essential oils containing (1S)-(ꢀ)-verbenone
have been reported to have antibacterial, acaricidal, and anti-
inflammatory properties,9 but the anti-ischemic effects of (1S)-
(ꢀ)-verbenone have not been elucidated yet. Verbenone is thought
to be naturally generated as either a biotransformation10 or an
auto-oxidation product of verbenol,8 which we previously identi-
fied as a lead compound with anti-ischemic and anti-inflammatory
effects.11 However, verbenol shows neither direct ROS/RNS
scavenging ability nor the ability to directly inhibit NMDA recep-
tor-coupled channel activity.11 In the present study, we report
synthesis and biological evaluation of novel verbenone derivatives
bearing modified styryl moieties, which are often found in natural
products with significant free radical scavenging and anti-inflam-
matory activity, such as curcumin, resveratrol, and ferulic acid.12
Commercially available (1S)-(ꢀ)-verbenone 1 was therefore
coupled with various benzaldehydes bearing protected hydroxy
moieties to afford verbenone derivatives 2a–i in the E-configura-
tion.13 Methoxymethyl groups of compounds 2a–i were then re-
moved by acid to afford verbenone derivatives 3a–i with
phenolic functionality along with alkoxyl or bromo substitutions
(Scheme 1).14
In the DPPH assay, most of the styryl derivatives with hydroxyl
groups 3a–f and 3h–i showed direct scavenging activities against
nitrogen free radicals (DPPHÅ) (Table 1). However, compound 3g
with a metha-hydroxyl group lacked DPPH radical scavenging
activity. As expected, a lack of hydroxyl moieties in the phenyl ring
such as compounds 4a–h and 5a–c resulted in no anti-oxidant
activity in the DPPH assay. Among the phenolic compounds exam-
ined, 3c and 3f with 3,4-dihydroxy groups showed better scaveng-
ing activity than vitamin C. In the ORAC assay, all derivatives 3a–i
containing phenolic groups showed higher peroxyl radical scav-
enging activity than trolox, a well-known anti-oxidant (Table 1).
Interestingly, compound 4e with a para-pyrrole group exhibited
significant peroxyl radical scavenging activity, even though it
lacked the phenolic moiety (Table 1). A pyrrole group can act as
a hydrogen atom transfer (HAT) agent alone and even better when
connected to the electron-supplying aryl group through stabiliza-
tion by resonance.15 However, this was not the case for compound
4e, since it is a pyrrole-1-yl derivative lacking a free H atom in the
N–H group. Compound 4e might demonstrate peroxyl radical scav-
enging activity in the ORAC assay as shown by other pyrrole N-con-
jugates with anti-oxidant activities.15
In order to assess the importance of the hydroxyl moieties on
styryl scaffold, (1S)-(ꢀ)-verbenone 1 was then coupled with vari-
ous benzaldehydes including methoxy and pyrrole groups in a sim-
ilar condition to afford compounds 4a–h (Scheme 2). It is
noteworthy to mention that the pyrrole and methoxy-substituted
phenyl groups may exhibit or modulate anti-oxidant activities
alone or when conjugated with other groups.15 In addition, a pyr-
idine moiety is often found in the pharmacophores of anti-oxidant
and anti-inflammatory reagents,16 or some classes of NMDA recep-
tor antagonists.17 Thus, we further synthesized pyridine deriva-
tives 5a–c (Scheme 2).
To evaluate anti-ischemic effects, we employed an in vitro
ischemic model with oxygen–glucose deprivation/reoxygenation
(OGD/R), which mimics a sudden disruption of blood supply and
the energy depletion occurring during ischemia.3 In cultured rat
cortical neurons, OGD-induced cell injury and loss of membrane
integrity increased the release of cytosolic lactate dehydrogenase
(LDH) into the media. Although compounds 3a–i showed signifi-
cant free radical scavenging activities, only three derivatives with
R1
R1
a
b
(88~100%)
(82~100%)
O
O
O
O
1
2
3
H
R1
1 = 4'-OMOM
2a
;
R1 = 4'-OH
3a;
R
R1 = 2'-OMe, 4'-OMOM
R1 = 2'-OMe, 4'-OH
3b
2b;
2c
2d
;
; R1 = 3',4'-diOMOM
R
R
1 = 3',4'-diOH
3c;
3d;
R1 = 3'-Br, 4'-OMOM
;
1 = 3'-Br, 4'-OH
R1 = 2',6'-diOMe, 4'-OMOM
R1 = 3',4'-diOMOM, 5'-OMe
R1 = 3'-OMOM
R1 = 2',6'-diOMe, 4'-OH
R1 = 3',4'-diOH, 5'-OMe
R1 = 3'-OH
3e
2e;
2f
;
3f;
;
2g
3g;
;
1 = 2'-OMOM
1 = 2'-OH
3h
2h;
;
R
R
R1 = 2'-OMOM, 4'-OMe
R1 = 2'-OH, 4'-OMe
2i
3i;
;
Scheme 1. Synthesis of compounds 3a–i. Reagents and conditions: (a) KOH, MeOH, 60 °C, 6 h; (b) 10% HCl, MeOH, rt, 24 h.
Z
Y
R1
a
b
X
(41~ 97%)
O
(77~ 87%)
O
O
O
O
H
4
1
H
5
R1
X
Z
Y
; R1 = H
; X = N, Y = C, Z = C
5b; X = C, Y = N, Z = C
5c
4a
5a
4b; R1 = 4'-F
; R1 = 4'-OMe
; R1 = 4'-Ph
; X = C, Y = C, Z = N
4c
4d
4e; R1 = 4'-pyrrol-1-yl
1 = 3',4'-diOMe
; R1 = 3',5'-diOMe
4h; R1 = 2',5'-diOMe
4f
;
R
4g
Scheme 2. Synthesis of compounds 4a–h and 5a–c. Reagents and conditions: (a) KOH, MeOH, 60 °C, 6 h; (b) NaOMe, MeOH, 60 °C, 6 h.

C. Ju et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5421–5425
5423
Table 1
Free radical scavenging activity of (1S)-(ꢀ)-verbenone derivatives
Compds
DPPH assay % inhibitiona
ORAC assay fold changeb
Compds
DPPH assay % inhibitiona
ORAC assay
fold changeb
1
ND
1.19 0.11
10.66 0.12
7.37 0.13
9.09 0.83
11.64 0.36
11.51 0.42
6.86 2.62
5.11 0.27
6.84 0.18
9.48 0.22
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
Trolox
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
—
1.92 0.05
1.81 0.09
2.64 0.14
1.85 0.09
6.81 0.03
2.73 0.12
3.14 0.04
2.25 0.11
1.63 0.05
1.59 0.04
1.56 0.02
3.11 0.07
3a
3b
3c
3d
3e
3f
3g
3h
3i
19.11 0.67
38.13 5.62
91.62 3.27
29.45 2.41
55.80 7.52
83.64 6.19
ND
4.64 0.60
51.21 3.83
Vit C
73.97 8.35
—
Data were expressed as mean SEM.
a
DPPH assay values were expressed as % inhibition in DPPH absorbance by derivatives at 80
M vitamin C (Vit. C). N = 9. ND: Not detected.
ORAC values were expressed as fold change of netAUC relative to vehicle control (14.44 0.83). N = 12.
lM, by comparison to the standard dose–response curves using from 0 to
500
l
b
phenolic groups (3a, 3c, and 3f) significantly reduced neuronal in-
jury (Fig. 1A), comparable to MK801, a well-known NMDA receptor
channel blocker. The tested compounds did not cause cytotoxicity
to cortical neurons (Fig. 1A).
In the H2DCF-DA assay, compounds 3c and 3f, but not 3a, sig-
nificantly decreased OGD-evoked intracellular oxidative stress
(Fig. 1B and C), which might contribute to their anti-ischemic ef-
fect. Compounds 3c and 3f, compared to 3a, showed better free
radical scavenging activities against nitrogen radicals from DPPH
(Table 1). Along with ROS, RNS is also elevated in ischemic regions
and plays a critical role in ischemic injury cascades.18 Inefficiency
of 3a in reducing intracellular oxidative stress might be partly
Figure 1. Neuroprotection by (1S)-(ꢀ)-verbenone derivatives against OGD/R-induced injury in an in vitro ischemic model. Cultured rat cortical neurons were exposed to OGD
(90 min) and subsequent reoxygenation (R, 9 h). Derivatives, MK801, or trolox (10 M or indicated concentrations of each) were pretreated for 30 min before OGD and
maintained during OGD/R; (A) Neuronal injury was assessed by the LDH assay. Data represent the mean SEM. N = 24; (B, C) H2DCFDA was loaded 1 h after reoxygenation
l
and intracellular oxidative stress was measured by an increase in fluorescence intensity (F.I.) after 2 h. Representative images (B, scale bar = 50 lm) and quantitative data; (C)
Data were expressed as the median (vertical column) and interquartile ranges from the first to the third quartile (vertical line). N = 9–18. ⁄P <0.05, ⁄⁄P <0.01, ⁄⁄⁄P <0.001:
versus OGD/R group.

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C. Ju et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5421–5425
Figure 2. The effect of (1S)-(ꢀ)-verbenone derivatives on NMDA-evoked excitotoxicity. (A) Cortical neurons were treated with 100
for 9 h. Derivatives or MK801 (10 M of each) administered 30 min before and maintained during NMDA treatment. Neuronal injury was assessed as % inhibition of LDH
release and expressed as the median (vertical column) and interquartile ranges (Q1–Q3, vertical line). N = 9–18; (B, C) Cells were loaded with Fluo-3 AM for 30 min in the
absence or presence of 10
M of 3f. 50 s after NMDA treatment, intracellular free calcium levels ([Ca2+]i) were assessed as the fold change in integrated fluorescence density of
Ca2+-bound Fluo-3 relative to basal levels. Representative images (B, scale bar = 50
m) and quantitative data; (C) Data were expressed as the median (vertical bar),
interquartile ranges (box), and min–max (whisker plots). N = 4–5. ⁄P <0.05, ⁄⁄P <0.01, ⁄⁄⁄P <0.001: versus NMDA-treated group.
lM NMDA for 15 min and then incubated
l
l
l
due to its limited scavenging activity against nitrogen radicals,
although further experiments are needed to confirm this
observation.
In addition to oxidative stress, excitotoxicity is a major injury
mechanism occurring during OGD and cerebral ischemia.19
Although none of these derivatives demonstrated anti-excitotoxic
activities comparable to MK801, derivatives 3a, 3c, 3f, 3i, and 5a
significantly reduced NMDA-evoked neuronal injury (Fig. 2A).
Moreover, treatment with 3f inhibited NMDA-induced intracellu-
lar calcium uptake (Fig. 2B and C).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.07.
038.
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Acknowledgments
This research was supported by the Korea Health Technology
R&D Project, the Ministry of Health & Welfare (#A100766 to W.-
K.K.) and by the Basic Science Research Program through the Na-
tional Research Foundation of Korea (NRF) funded by the Ministry
of Education, Science and Technology (#2011-0026325 to Y.C.),
Republic of Korea. We are grateful to Dr. In-Young Choi for helpful
scientific information regarding (S)-cis-verbenol and Dr. Paul L.
Prather (University of Arkansas) for careful reading.
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C. Ju et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5421–5425
5425
14. Procedure for the synthesis of (1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-
dimethylbicyclo[3.1.1]hept-3-en-2-one (3f). For the synthesis of diene from
301.1440, found 301.1453; HPLC analysis: flow rate of 0.2 mL/min, 90%
acetonitrile in water in 20 min) 100% (tR = 3.46 min).
(1S)-(ꢀ)-verbenone by aldol condensation, (1S)-(ꢀ)-verbenone
1
(200 mg,
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benzaldehyde (332 mg, 2.00 mmol) while stirring in MeOH (7 mL) and
treated with KOH (149 mg, 2.66 mmol). The mixture was stirred at 60 °C for
6 h and cooled to room temperature. After a few drops of H2O were added, the
mixture was allowed to stand for 24 h at ambient temperature. The mixture
was concentrated in vacuo and purified by column chromatography toafford 2f
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as
a yellow syrup (465 mg, 90%). To a stirred solution of 2f (200 mg,
0.51 mmol) in MeOH (3 mL), 10% HCl was added dropwise. The reaction
mixture was then stirred overnight until the completion of reaction. After
addition of saturated NaHCO3, the mixture was extracted with ethyl acetate
and dried over anhydrous MgSO4. The final compound was separated by
column chromatography on a silica gel to give 3f as a yellow solid (142 mg,
92%): mp 168–170 °C; ½a D20
ꢁ
ꢀ158.8° (c 1.0, MeOH); 1H NMR (CDCl3, 500 MHz) d
6.82 (d, J = 16.50 Hz, 1H), 6.79 (d, J = 1.71 Hz, 1H), 6.79 (d, J = 16.50 Hz, 1H),
6.65 (d, J = 1.71 Hz, 1H), 5.91 (s, 1H), 5.71 (br s, 2H), 3.93 (s, 3H), 3.09 (t, J = 5.75
Hz, 1H), 2.91 (td, J = 5.59, 9.35 Hz, 1H), 2.73 (dt, J = 1.71, 5.75 Hz, 1H), 2.12 (d,
J = 9.54 Hz, 1H), 1.58 (s, 3H), 1.02 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 204.92,
165.07, 147.23, 144.24, 135.54, 134.13, 128.07, 125.50, 121.62, 108.63, 58.08,
56.25, 53.12, 43.75, 40.18, 26.78, 22.16; HRMS calculated for C18H20O4 (M+H)