Natural products inspired synthesis of neuroprotective agents against H2O2-induced cell death

Article abstract of DOI:10.1016/j.bmcl.2013.01.013

Stroke is a debilitating disease and the third leading cause of death in the USA, where over 2000 new stroke cases are diagnosed every day. Treatment options for stroke-related brain damage are very limited and there is an urgent need for effective neurop

Full text of DOI:10.1016/j.bmcl.2013.01.013

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1232–1237
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Natural products inspired synthesis of neuroprotective agents
against H2O2-induced cell death
a,b,
Jehad Almaliti a, Shadia E. Nada a, Bryaune Carter a, Zahoor A. Shah a, , L. M. Viranga Tillekeratne
⇑
⇑
a Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, Toledo, OH 43606, USA
b Department of Chemistry, College of Natural Sciences and Mathematics, The University of Toledo, Toledo, OH 43606, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Stroke is a debilitating disease and the third leading cause of death in the USA, where over 2000 new
stroke cases are diagnosed every day. Treatment options for stroke-related brain damage are very limited
and there is an urgent need for effective neuroprotective agents to treat these conditions. Comparison of
the structures of several classes of neuroprotective natural products such as limonoids and cardiac gly-
cosides revealed the presence of a common structural motif which may account for their observed neu-
roprotective activity. Several natural product mimics that incorporate this shared structural motif were
synthesized and were found to possess significant neuroprotective activity. These compounds enhanced
cell viability against H2O2 induced oxidative stress or cell death in PC12 neuronal cells. The compounds
were also found to enhance and modulate Na+/K+-ATPase activity of PC12 cells, which may suggest that
the observed neuroprotective activity is mediated, at least partly, through interaction with Na+/K+-
ATPase.
Received 20 November 2012
Revised 27 December 2012
Accepted 2 January 2013
Available online 11 January 2013
Keywords:
Neuroprotective agents
Na+/K+-ATPase pump
Stroke
Oxidative damage
Natural products
Ó 2013 Elsevier Ltd. All rights reserved.
Stroke is a major health concern in the industrialized countries.
It is the third leading cause of death in the USA, ranking after heart
disease and cancer.1 It is also the leading cause of long term dis-
ability, resulting in enormous financial burden on affected families
and on the health care system. Stroke-related healthcare costs are
rising, with nearly 1 million hospitalizations each year in the U.S.
and an estimated direct medical cost of 25.2 billion for the year
2007.2 Currently available treatment options for stroke-related
brain damage are limited. The only FDA approved drug for acute
ischemic stroke, IV recombinant tissue plasminogen activator (rt-
PA), unfortunately can be received by only 5–10% of acute ischemic
stroke (AIS) patients because of the less than 3 h restrictive thera-
peutic time window within which it must be administered.3 There-
fore, there is an urgent need for identification of new
neuroprotective drug candidates and drug targets for treatment
of ischemic stroke.
Natural products are a prolific source of bioactive agents of di-
verse structure and varying biological activity. They are the single
most productive source of lead molecules for development as clin-
ically useful drugs for human disorders. In the search for neuropro-
tective agents from natural sources, a number of plant extracts and
several natural products isolated from them have been reported to
provide neuroprotection against ischemic stroke.4–6 In addition, a
wide range of natural product derivatives and natural product
mimics of synthetic origin such as estrogen-like compounds,7 kav-
alactone derivatives,8 glucose-containing flavones,9,10 arylidene-
pregnenolone derivatives,11 lignophenol derivatives,12 triazine
derivatives,13 pyrazolyl-2,4-thiazolidinedione derivatives,14 phe-
nolic thiazoles,15 and indole derivatives16 have been shown to pro-
tect neurons against oxidative damage induced by H2O2 or by other
oxidative stress conditions. Among neuroprotective natural prod-
ucts, a group of naturally occurring limonoids dictamnusine 1, dic-
tamdiol 2, fraxinellone 3, calodendrolide 4, obacunone 5 and
limonin 6 (Fig. 1) isolated from Dictamnus dasycarpus were re-
ported to have significant neuroprotective activity against gluta-
mate-induced neurotoxicity in rat cortical cells.5 Obacunone 5
was also reported to be protective against glutamate-induced oxi-
dative damage in mouse hippocampal HT22 cells.17 Further, it in-
duced cell resistance to oxidative injury from glutamate-induced
cytotoxicity in HT22 cells, presumably mediated through p38
MAPK pathway-dependent heme oxygenase-1 expression.17
The cardiac glycoside neriifolin 7 isolated from the yellow ole-
ander Thevetia neriifolia and its 16-b-acetoxy derivative oleandrin,
Abbreviations: IV, intravenous; FDA, food and drug administration; AIS, acute
ischemic stroke; rt-PA, recombinant tissue plasminogen activator; NMR, nuclear
magnetic resonance; NOE, nuclear overhauser effect; m-CPBA, meta-chloroperoxy-
benzoic acid; ORTEP, oak ridge thermal ellipsoid plot; ROS, reactive oxygen species;
GSH, glutathione; Pi, inorganic phosphate; TLC, thin layer chromatography; HRMS,
high resolution mass spectrometry.
⇑
Corresponding authors. Tel.: +1 419 383 1587; fax: +1 419 530 7946 (Z.A.S.);
tel.: +1 419 530 1983; fax: +1 419 530 7946 (L.M.V.T.).
E-mail addresses: Zahoor.Shah@utoledo.edu (Z.A. Shah), ltillek@utnet.utoledo.e-
du (L.M. Viranga Tillekeratne).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.013

J. Almaliti et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1232–1237
1233
O
O
O
O
O
O
O
O
β
-Glu
O
O
HO
O
O
O
O
OH
1
3
4
2
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
5
6
Figure 1. Neuroprotective limonoids.
O
O
R5
R4
R3
R2
OH
R1
O
R6
OH
H3C
H3C
O
O
O
HO
Me
HO
MeO
O
7
R1
=
R1
=
8
O
O
OH
H3C
OH
OH
2 = R3 = R4 = R5 = R6 = H
2 = R3 = R4 = R5 = R6 = H
R
R
OH
H3C
H3C
O
Me
HO
R1
10
=
O
R1
=
HO
O
O
9
O
O
HO
H3C
OH
OH
OH
R2 = R3 = R4 = R6 = H; R5 = OH
R
2 = R3 = R4 = R5 = R6 = OH
Figure 2. Neuroprotective cardiac glycosides.
and to a lesser extent the glycosides digitoxin 8, digoxin 9 and oua-
bain 10 (Fig. 2), provided protection against neuronal cell death in-
duced by oxygen and glucose deprivation in a brain slice assay, an
effect believed to be mediated through their putative target the
Na+/K+-ATPase.6,18 Sublethal concentrations of ouabain has been
shown to have neuroprotective activity against excitotoxicity med-
iated neuronal injury and this effect has been attributed to intra-
cellular cascades linked to ouabain interaction with Na+/K+-
ATPase leading to modulation of subcellular Bcl-2 levels.19 How-
ever, cardiac glycosides are substrates for P-glycoprotein, which
precludes their usefulness as therapeutics for ischemic stroke, as
effective penetration of the blood brain barrier to build up thera-
peutic levels of the drugs within the CNS is inhibited.6 Neverthe-
less, they may serve as lead molecules for designing more
effective drug candidates.
R
O
furanyl
R =
Me
O
O
γ
-lactone
R =
Y
X
O
X = CO or -C-CO
Y = O or CH2
11
Figure 3. Structural motif shared by neuroprotective natural products.
or c-lactone) side chain and with or without an oxygen function
at the ring junction (Fig. 3). Intrigued by the presence of this com-
mon structural motif in neuroprotective natural products, we de-
signed and synthesized several structural congeners of this unit
to explore their neuroprotective properties. The molecules de-
signed for synthesis are shown in Figure 4.
A comparison of the structures of these naturally occurring
neuroprotective molecules revealed that they share a common
bicyclic ring system with a five-membered heterocycle (furanyl

1234
J. Almaliti et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1232–1237
O
O
O
O
O
O
H
H
O
O
OH
OH
H
OH
14
OH
OH
O
O
O
O
O
12
13
15
16
17
Figure 4. Target molecules designed for synthesis.
O
O
21
OHC
a) Ac2O,
ZnCl2, ZnI2
+
b) -TsOH
p
NaOH, 65%
19
20
(major)
22
18
O
O
20 19
80% (6:1,
:
)
O
(minor)
O
O
O
NOE
NOE
HCO2H, H3PO4
70%
NaH, (EtO)3P
O2, 65%
NEt3
MeI
99%
H
OH
O
O
O
O
13
23
12
H2, Pd/C
60%
1. TES-OTf, NEt3
2.
m
-CPBA
61%
O
NOE
O
H
O
O
OH
15
OH
O
14
Scheme 1. Synthesis of target compounds 12–15. Inset: ORTEP drawing derived from the single-crystal X-ray analysis of racemic 14. The crystal structure of compound 14
has been deposited at the Cambridge Crystallographic Data Centre and allocated the deposition number CCDC 871250.
The synthesis of target molecules 12, 13, 14 and 15 is shown in
Scheme 1. Lewis acid-catalyzed acetylation of 1-methylcyclohex-
ene 18 gave a nearly 1:1 mixture of the two ketones 19 and 20.
Acid-catalyzed isomerization using p-toluenesulfonic acid in ben-
zene followed by bulb-to-bulb distillation of the product gave a
configuration of the newly generated stereo center being con-
firmed by an NOE observed between its H atom and the angular
methyl group.
A similar synthetic approach using 2-furfuraldehyde 24 instead
of 3-furfuraldehyde 21 gave the target compounds 16 and 17
(Scheme 2). The configuration at the newly generated stereo center
in 17 was confirmed by NOE experiment as before. The 1H NMR,
13C NMR and HRMS data of all the compounds synthesized were
in full agreement with the proposed structures.36
Oxidative damage mediated by reactive oxygen species (ROS)
has been implicated as a major cause of cellular injuries in a vast
variety of clinical abnormalities including cancer, diabetes, aging,
cardiovascular disease, and neurodegenerative disorders. Reactive
oxygen intermediates are produced in all mammalian cells, partly
as a result of normal cellular metabolism and partly due to activa-
tion of a variety of oxidant-producing enzymes in response to
exogenous stimuli. Excessive accumulation of reactive oxidants
and the intracellular level of reactive oxidants are toxic.24 It is well
established that treatment of PC12 cells with H2O2 results in nucle-
ar damage, decrease in mitochondrial transmembrane potential,
cytosolic accumulation of cytochrome-c, activation of caspase-3,
increase in the formation of reactive oxygen species. (ROS) and
depletion of glutathione (GSH).25 Protection of PC12 cells against
mixture of the two ketones in which the
a,b-unsaturated ketone
20 was the major isomer (19:20: ꢀ1:6 by 1H NMR).20 No attempt
was made to separate the mixture and it was subjected to aldol
condensation with 3-furfuraldehyde 21. Chromatography separa-
tion gave the divinylketone 22. It was converted to 12, consisting
of the bicyclic motif of our interest, by acid-catalyzed Nazarov
cyclization21 using a mixture of formic acid and phosphoric acid.
Rubottom oxidation22 of 12 with triethylsilyl triflate and triethyl-
amine followed by m-CPBA to obtain
resulted in mainly the Baeyer–Villiger oxidation product lactone
14 with low yields of 13. However, -peroxidation via the enolate,
a-hydroxylation product 13
a
followed by reduction with triethyl phosphate gave quantitative
yields of the cis-fused alcohol 13.23 The cis configuration of 13
was confirmed by conversion to the methyl ether 23 in which an
NOE was observed between the angular methyl group and the
methyl ether group. The cis-configuration of 14 was confirmed by
single crystal X-ray crystallography (Scheme 1). Catalytic hydroge-
nation of the
a,b-unsaturated ketone 13 gave the ketone 15, the

J. Almaliti et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1232–1237
1235
O
O
O
24
HCO2H, H3PO4
39%
OHC
NaOH, 65%
H
26
O
O
O
20
25
NOE
O
O
H
H2, Pd/C
70%
NaH, (EtO)3P
O2, 70%
OH
OH
O
O
16
17
Scheme 2. Synthesis of target compounds 16 and 17.
Figure 5. Neuroprotective activity of the test compounds. Experiments were repeated in triplicates with three separate batches of cultures. Control—naïve or no treatment;
H2O2—cells treated with 100 M H2O2 only; 12–17—cells pretreated with different concentrations of test compounds (12, 13, 14, 15, 16 and 17) for 6 h followed by 18 h
exposure to 100 M H2O2. At the end of the treatment, cells were harvested for cell viability assay. Data are presented as means SEM. Student t-test was performed for
statistical comparison between control and other treatments. A value of p <0.05 was considered to be statistically significant. #versus control and ⁄versus H2O2.
l
l
H2O2 induced cell death can therefore be used to measure the neu-
roprotective activity of test agents. In order to test the effects of
these compounds on the viability of PC12 cells against H2O2 in-
duced cell death, cultured cells were pretreated with different con-
protective potential with increasing concentration in terms of cell
viability, which gradually increased and started plateauing at
150 lM (Fig. 5). Lowest concentrations of compounds 14 and 17
were not as potent as compound 12 and 13, nevertheless showed
centrations (0.1, 1, 10, 50, 100 and 150
before adding 100 M of H2O2 for additional 18 h of incubation.
Treating cells with 100 M of H2O2 alone induced profound cell
death compared to control (Fig. 5). Control cells did not receive
any treatment, therefore, 100% cell survival was observed. Cells
l
M) of test compounds 6 h
a pattern of increasing protection with increasing concentration
with 17 plateauing at 150 lM concentration. These results suggest
l
l
that all these compounds, especially compounds 12 and 13 have
neuroprotective properties and may have potential to be devel-
oped as neuroprotective agents. It is interesting to note that both
compounds 12 and 13 possess furan-3-yl moieties attached to
the bicyclic system, as are commonly found in naturally occurring
limonoids. Corresponding analogue 16 with a furan-2-yl moiety is
less active at lower concentrations. Although compounds 14 and
15 contain a furan-3-yl moiety, they lack an unsaturated cyclo-
pentenone ring as a part of the bicyclic system. These results indi-
cate that hexahydro-1H-ineden-1-one structural motifs with a
pretreated with concentrations 0.1–150 lM of all compounds sig-
nificantly (p <0.0001) enhanced cell viability and reversed the H2O2
induced oxidative stress. Compounds 15, and 16 did not show a
pattern of increased protection with increasing concentrations
(0.1–150
observed to have the highest protection at the lowest concentra-
tions of 0.1 and 10 M and also showed a pattern of increasing
lM) in terms of cell viability. Compounds 12 and 13 were
l

1236
J. Almaliti et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1232–1237
Figure 6. Effect of compounds on Na+/K+-ATPase activity: Experiments were repeated in triplicate. Control—naïve or no treatment; H2O2—cells treated with 100
lM H2O2
only; 12–17, cells co-treated with 50 lM of test compounds (12, 13, 14, 15, 16 and 17) and 100 l
M of H2O2 for 1 h. At the end of the treatment, cells were harvested for Na+/
K+-ATPase assay. Data are presented as means SEM. Student t-test was performed for statistical comparison between control and other treatments. A value of p <0.05 was
considered to be statistically significant. #versus control and ⁄versus H2O2.
furan-3-yl moiety attached to C3 position and with or without a
hydroxyl function at the ring juncture possess significant neuro-
protective properties and are potential lead molecules to be devel-
oped as neuroprotective agents.
considered to be endogenous or basal activity. The results are
shown in Figure 6. A significant reduction in Na+/K+-ATPase activ-
ity was observed in the presence of 100 lM H2O2 in comparison to
control (p <0.02). Treatment with compounds only (without H2O2)
did not show any significant effects on the Na+/K+-ATPase activity
as compared to control. However, when the cells were challenged
The neuroprotective properties of cardiac glycosides are be-
lieved to be mediated through interaction with the Na+/K+-ATPase
pump.6,18,19,26 Na+/K+-ATPase is an ATP-powered ion pump that
establishes concentration gradients for Na+ and K+ ions across the
plasma membrane in all animal cells by pumping Na+ from the
cytoplasm into the outside of cell and K+ from the extracellular
medium into the cell.27,28 Blocking the Na+/K+-ATPase pump may
lead to apoptosis. Neurodegeneration upon administration of oua-
bain has been attributed to inhibition of Na+/K+-ATPase
pump.19,29,30 However, ouabain has been shown to be able to initi-
ate cell proliferation or cell death in a dose-dependent manner,31
with non-toxic concentrations of it being neuroprotective.19 Suble-
thal concentrations of ouabain have been shown to provide neuro-
protection against excitotoxicity mediated neuronal injury through
interaction with Na+/K+-ATPase leading to modulation of subcellu-
lar Bcl-2 levels.19 Other studies have shown that restoring Na+/K+-
ATPase activity protect cells against oxidative cell damage.32,33 We
therefore, tested the compounds for their effect on the activity on
Na+/K+-ATPase pump by direct method using adherent PC12 cells
as described in previous studies,34,35 with some modifications.
The assay was run in two parallel steps; one for the evaluation of
total ATPase activity and the other for the evaluation of ouabain
insensitive ATPase activity. ATPase activity was calculated by col-
orimetric quantification of inorganic phosphate (Pi) formed in
30 min, and Na+/K+-ATPase activity was determined by the differ-
ence between the results in the absence and presence of ouabain.
In order to test the effects of these compounds on Na+/K+-ATPase
with 100 l
M H2O2 in the presence of test compounds, Na+/K+-ATP-
ase activity was observed to be significantly restored with com-
pound 12, (p <0.014), compound 14 (p <0.006), compound 16 (p
<0.003) and compound 17 (p <0.002). No direct correlation could
be drawn between structural variations and the variations in
Na+/K+-ATPase activity. However, the results may suggest that
the Na+/K+-ATPase enhancing and modulating effects of these com-
pounds may be responsible, at least in part, for their observed neu-
roprotective activity. Since, Na+/K+-ATPase has a significant role in
cerebral ischemia pathophysiology, developing such compounds
with Na+/K+-ATPase enhancing activity would be an ideal proposi-
tion for drug discovery in the treatment of neurologic disorders
such as stroke and cerebral ischemia. Further studies are contin-
uing to elucidate the mechanism(s) of action and the putative tar-
get of these molecules.
In summary, we report the design and synthesis of several nat-
ural product mimics that incorporate a common structural unit
shared by several classes of natural products with neuroprotective
activity. Neuronal viability was restored with different concentra-
tions of these compounds against H2O2 induced oxidative stress.
Compounds with hexahydro-1H-ineden-1-one structural motifs
with a furan-3-yl moiety attached to C3 position and with or with-
out a hydroxyl function at the ring juncture in particular possessed
significant neuroprotective properties and may be useful as lead
molecules for developing neuroprotective agents.
pump, PC12 cells were treated with each compound at 50
l
M in
Oxidative stress is an important factor in the initiation and pro-
gression of many neurodegenerative diseases. The results suggest
that enhancing and modulating effects on Na+/K+-ATPase activity
may be responsible, at least in part, for the neuroprotective
the presence and absence of 100 lM H2O2 for one hour prior to
the Na+/K+-ATPase assay. Control cells did not receive any treat-
ment and the Na+/K+-ATPase activity observed in these cells was

J. Almaliti et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1232–1237
1237
properties of these compounds. Na+/K+-ATPase has a significant
role in cerebral ischemia pathophysiology. Therefore, compounds
with Na+/K+-ATPase enhancing activity will be useful as agents in
the treatment of neurologic disorders such as stroke and cerebral
ischemia. Further studies are in progress to validate their efficacy
as potential lead molecules for developing therapeutic agents for
neurodegenerative diseases.
18. Wang, J. K.; Portbury, S.; Thomas, M. B.; Barney, S.; Ricca, D. J.; Morris, D. L.;
Warner, D. S.; Lo, D. C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10461.
19. Golden, W. C.; Martin, L. J. Neuroscience 2006, 137, 133.
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1998, 54, 875.
21. Fernandez Mateos, A.; Martin de la Nava, E. M.; Rubio Gonzalez, R. Tetrahedron
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22. Hassner, A.; Reuss, R. H.; Pinnick, H. W. J. Org. Chem. 1975, 40, 3427.
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Acknowledgements
27. Albers, R. W. Annu. Rev. Biochem. 1967, 36, 727.
This work was supported partly by a Grant from the National
Institutes of Health (R00AT004197) to Z.A.S. The authors thank
Dr. Kristin Kirschbaum and Nicholas Amato for X-ray crystallogra-
phy analysis.
28. Post, R. L.; Hegyvary, C.; Kume, S. J. Biol. Chem. 1972, 247, 6530.
29. Stelmashook, E. V.; Weih, M.; Zorov, D.; Victorov, I.; Dirnagl, U.; Isaev, N. FEBS
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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.
01.013.
35. Xie, Z. J.; Wang, Y. H.; Ganjeizadeh, M.; McGee, R., Jr.; Askari, A. Anal. Biochem.
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36. Compound characterization data:
Compound 12: Yellow oil, mp 70–73 °C. 1H NMR (400 MHz, CDCl3) 1.19–1.25
(m, 1H), 1.4–1.57 (m, 2H), 1.65–1.68 (m, 1H), 1.43 (s, 3H), 1.91–1.94 (m, 1H),
2.09–2.14 (m, 1H), 2.21–2.24 (m, 1H), 6.18 (s, 1H), 6.60 (m, 1H), 7.48 (m, 1H),
7.80 (s, 1H). 13C NMR (CDCl3, 100 MHz): 20.17, 21.38, 21.63, 24.97, 36.76,
45.73, 55.18, 110.03, 120.17, 125.82, 142.67, 143.89, 173.96, 208.96 ppm.
HRMS: (ESI) calcd for C14H16O2 [M+Na]+ 239.1048; found 239.1056.
Compound 13: White crystals, mp 74–80 °C. 1H NMR (400 MHz, CDCl3) 1.25–
1.31(m, 1H),1.32 (s, 1H), 1.44–1.49 (m, 1H), 1.65–1.77 (m, 5H), 1.96–2.02 (m,
1H), 2.59 (s, 1H), 6.19 (s, 1H), 6.62 (d, J = 1.2 Hz, 1H), 7.51 (m, 1H), 7.85 (s, 1H).
13C NMR (CDCl3, 100 MHz): d 19.56, 20.83, 26.18, 32.83, 33.93, 50.20, 80.98,
109.95, 120.30, 123.25, 142.85, 144.10, 172.75, 210.19 ppm. HRMS: (ESI) calcd
for C14H16O2 [M+Na]+ 255.0998; found 255.0992.
References and notes
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Compound 14: White crystals, mp 110–112 °C. 1H NMR (400 MHz, CDCl3) 1.35
(s, 3H), 1.48–1.49 (m, 2H), 1.71–1.72 (m, 3H), 1.88–2.02 (m, 3H), 3.40 (br, 1H),
6.05 (s, 1H), 6.51–6.52 (m, 1H), 7.46 (s, 1H), 7.61 (s, 1H). 13C NMR (CDCl3,
100 MHz): 20.02, 20.59, 22.72, 34.98, 36.89, 42.91, 104.80, 110.44, 115.93,
122.33, 141.46, 143.68, 157.49, 164.28 ppm. HRMS: (ESI) calcd for C14H16O2
[M+Na]+ 271.0946; found 271.0936.
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Compound 15: White solid, mp 70–73 °C. 1H NMR (400 MHz, CDCl3) 0.96–1.04
(m, 1H), 1.13 (s, 3H), 1.22–1.25 (m, 2H), 1.42–1.47 (m, 1H), 1.59–1.67 (m, 2H),
1.98–2.04 (m, 1H), 2.38–2.46 (m, 1H), 2.74–2.81 (m, 1H), 3.46–3.51 (t,
J = 9.6 Hz, 1H), 6.29 (s, 1H), 7.26 (s, 1H), 7.39 (m, 1H). 13C NMR (CDCl3,
100 MHz): 16.51, 20.81, 23.74, 28.73, 31.39, 38.55, 38.88, 45.01, 80.37, 111.12,
122.63, 140.24, 143.03, 215.70 ppm. HRMS: (ESI) calcd for C14H16O2 [M+Na]+
257.1154; found 257.1154.
Compound 16: White crystals, mp 76–81 °C. 1H NMR (400 MHz, CDCl3) 1.24–
1.35 (m,1H),1.36 (s, 3H) 1.42–1.49 (m, 1H), 1.59–1.83 (m, 5H), 2.07–1.13
(m,1H), 2.55 (s,1H), 6.36 (s,1H), 6.56 (m, 1H), 6.90 (d, J = 3.66 Hz, 1H), 7.59 (d,
J = 1.83 Hz, 1H). 13C NMR (CDCl3, 100 MHz): d 19.28, 20.51, 25.73, 32.80, 33.73,
49.22, 80.73, 112.81, 115.51, 121.06, 145.57, 149.33, 167.45, 209.85 ppm.
HRMS: (ESI) calcd for C14H16O2 [M+Na]+ 255.0998; found 255.0997.
Compound 17: White solid, mp 68–72 °C. 1H NMR (400 MHz, CDCl3) 1.01–1.09
(m, 2H), 1.23 (s, 3H), 1.44–1.45 (m, 2H), 1.57–1.67 (m, 3H), 1.99–2.03 (m, 1H),
2.66–2.83 (m, 2H), 3.61–3.66 (t, J = 8.0 MHz, 1H), 6.11 (d, J = 1.2 Hz, 1H), 6.33–
6.34 (m, 1H), 7.37 (s,1H). 13C NMR (CDCl3, 100 MHz): d 16.68, 20.79, 23.67,
28.61, 31.50, 37.05, 41.59, 45.83, 80.35, 107.24, 110.30, 141.80, 153.96,
215.14 ppm. HRMS: (ESI) calcd for
257.1147.
C
14H16O2 [M+Na]+ 257.1154; found
16. Mohareb, R. M.; Ahmed, H. H.; Elmegeed, G. A.; Abd-Elhalim, M. M.; Shafic, R.
W. Bioorg. Med. Chem. 2011, 19, 2966.
17. Jeong, G. S.; Byun, E.; Li, B.; Lee, D. S.; An, R. B.; Kim, Y. C. Arch. Pharmacol. Res.
2010, 33, 1269.