DL-3-n-butylphthalide-Edaravone hybrids as novel dual inhibitors of amyloid-β aggregation and monoamine oxidases with high antioxidant potency for Alzheimer's therapy

Article abstract of DOI:10.1016/j.bmcl.2017.01.050

Considering the complex etiology of Alzheimer's disease (AD), multifunctional agents may be beneficial for the treatment of this disease. A series of DL-3-n-butylphthalide-Edaravone hybrids were designed, synthesized and evaluated as novel dual inhibitors of amyloid-β aggregation and monoamine oxidases. Among them, compounds 9a–d exhibited good inhibition of self-induced Aβ_1-42aggregation with inhibition ratio 57.7–71.5%. For MAO, these new hybrids exhibited good balance of inhibition for MAO-A and MAO-B. In addition, all target compounds retained the antioxidant activity of edaravone, showed equal or better antioxidant activity than edaravone. The results of the parallel artificial membrane permeability assay for blood-brain barrier indicated that compounds 9a–d would be able to cross the blood-brain barrier and reach their biological targets in the central nervous system. The promising results in all assays demonstrated that the strategy behind the designing of compounds was rational and favourable. Taken together, these preliminary findings suggested that the compounds with the strongest bioactivity deserves further investigated for pharmacological development in AD therapy.

Full text of DOI:10.1016/j.bmcl.2017.01.050

Accepted Manuscript
DL-3-n-butylphthalide-Edaravone hybrids as novel dual inhibitors of amyloid-
β aggregation and monoamine oxidases with high antioxidant potency for
Alzheimer’s therapy
Xiaoming Qiang, Yan Li, Xia Yang, Li Luo, Rui Xu, Yunxiaozhu Zheng,
Zhongcheng Cao, Zhenghuai Tan, Yong Deng
PII:
S0960-894X(17)30063-X
http://dx.doi.org/10.1016/j.bmcl.2017.01.050
BMCL 24626
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 October 2016
6 December 2016
14 January 2017
Please cite this article as: Qiang, X., Li, Y., Yang, X., Luo, L., Xu, R., Zheng, Y., Cao, Z., Tan, Z., Deng, Y.,
DL-3-n-butylphthalide-Edaravone hybrids as novel dual inhibitors of amyloid-β aggregation and monoamine
oxidases with high antioxidant potency for Alzheimer’s therapy, Bioorganic & Medicinal Chemistry Letters (2017),
doi: http://dx.doi.org/10.1016/j.bmcl.2017.01.050
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DL-3-n-butylphthalide-Edaravone hybrids as novel dual inhibitors of amyloid-β
aggregation and monoamine oxidases with high antioxidant potency for
Alzheimer’s therapy
Xiaoming Qianga, Yan Lia, Xia Yanga, Li Luoa, Rui Xua, Yunxiaozhu Zhenga, Zhongcheng Caoa,
Zhenghuai Tanb,* and Yong Denga,*
aDepartment of Medicinal Chemistry, Key Laboratory of Drug Targeting and Drug Delivery System
of the Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu, 610041,
China
bInstitute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan academy of
Chinese Medicine Sciences, Chengdu, 610041, China
* Corresponding Author.
Phone, +86-28-85258982; E-mail: tanzhh616@163.com (Zhenghuai Tan)
Phone, +86-28-85503790; E-mail: dengyong@scu.edu.cn (Yong Deng)
1

Abstract
Considering the complex etiology of Alzheimer’s disease (AD), multifunctional agents may be
beneficial for the treatment of this disease. A series of DL-3-n-butylphthalide-Edaravone hybrids
were designed, synthesized and evaluated as novel dual inhibitors of amyloid-β aggregation and
monoamine oxidases. Among them, compounds 9a-d exhibited good inhibition of self-induced Aβ1-42
aggregation with inhibition ratio 57.7%-71.5%. For MAO, these new hybrids exhibited good balance
of inhibition for MAO-A and MAO-B. In addition, all target compounds retained the antioxidant
activity of edaravone, showed equal or better antioxidant activity than edaravone. The results of the
parallel artificial membrane permeability assay for blood-brain barrier indicated that compounds
9a-d would be able to cross the blood-brain barrier and reach their biological targets in the central
nervous system. The promising results in all assays demonstrated that the strategy behind the
designing of compounds was rational and favourable. Taken together, these preliminary findings
suggested that the compounds with the strongest bioactivity deserves further investigated for
pharmacological development in AD therapy.
Keywords: Alzheimer’s disease; DL-NBP-Edaravone hybrids; Aβ aggregation inhibitors, MAO
inhibitors; antioxidant.
2

Alzheimer’s disease (AD) is an age-related neurodegenerative process characterized by a
progressive memory loss, decline in language skills and other cognitive impairments.1 Although
several factors including massive loss of cholinergic neurons, amyloid-β aggregate deposition,
neurofibrillary tangles, oxidative stress and dyshomeostasis of biometals have been demonstrated to
play significant roles in the pathogenesis of AD, the exact etiology is still not fully understood.2,3
Among the multiple factors that induce AD, Aβ plays a primary role. The aggregation of Aβ
leads to the formation of senile plaques, resulting in neuronal dysfunction in AD patients.4 Moreover,
the progressive accumulation of Aβ is accompanied by oxidatives stress and inflammation, which
leads to neurodegeneration. Therefore, inhibition of Aβ aggregation is currently being considered as
a potential target for AD therapy.5
Recently, monoamine oxidases (MAOs), the important factors involved in oxidative stress, have
attracted more and more attention. MAOs are flavin adenine dinucleotide (FAD) containing enzymes
that catalyze the deamination of endogenous and xenobiotic amines and are responsible for the
regulation and metabolism of major monoamine neurotransmitters.6 MAOs have two isoforms
named MAO-A and MAO-B.7 MAO-A preferentially metabolizes of serotonin, adrenaline, and
noradrenaline. While MAO-B accelerates the oxidative deamination of β-phenethylamine and
benzylamine, resulting in exacerbated production of free radicals thus causing oxidative stess.8
Tyramine is a substrate for both MAO-A and MAO-B. Traditional non-selective MAO inhibitors
could inhibit the first-pass metabolism of exogenous tyramine. Treatment with non-selective MAO
inhibitors may result in the accumulaion of tyramine and have the potential to precipitate a dangerous
hypertensive crisis, the so-called “cheese effect”. The selective MAO-B inhibitors and the selective
and reversible inhibitors of MAO-A are free from this potential interaction.9 Presently, selective
inhibitors of MAO-A have been shown to be effective antidepressants, while selective MAO-B
3

inhibitors are useful in neurodegenerative disease such as AD.10 Moreover, AD patients commonly
have depressive symptoms which have been considered as risk factors for the development of AD.11
Therefore, it is resonable to expect that selective MAO inhibitors could contribute to senile
depression and AD therapy. On the other hand, favourable results have indicated that MAO increases
the expression of β-secretase and γ-secretase and improves Aβ generation from APP (amyloid
precursor protein).12 Additionally, MAO could be also associated with formation of neurofibrillary
tangles. Thus, MAO inhibition is included in mechanisms of action of multi-target drugs, newly
developed to treat AD.13
Oxidative stress represents one of the key processes in AD pathogenesis, related to formation of
both amyloid plaques and neurofibrillary tangles as well as to alteration of many biomolecules.14
There are indeed several proofs indicated that not only can Aβ induce oxidative stress but its
generation is also increased as a consequence of oxidative stress, thus forming a vicious circle.15
Therefore, the successful protection of neuronal cells from oxidative damage could potentially
prevent AD.
Due to the complexity of AD, drugs that modulate the activity of a single target might not be
sufficient to alter the progression of this disease. Thus, a more appropriate therpeutic strategy based
on the multi-target-directed ligands (MTDLs) paradigm has recently been proposed and applied to
design multi-target compounds against AD.16 Recently, our work is focused on the design of new
multifunctional molecules endowed with MAO inhibitory activities, inhibition of Aβ aggregation
properties, and antioxidant activity, by connecting molecules with such properties.
DL-3-n-butylphthalide (DL-NBP) was synthesized and received approval by the State Food and
Drug Administration of China for clinical use in stroke patients in 2002.17 It is a multi-target
neuroprotective agent, significantly improving mitochondrial function, decreasing neuronal apoptosis,
4

reducing oxidative damages and inhibiting inflammation.18 In addition, Wang’s group found that
DL-NBP improved the cognitive impairment and decreased Aβ-induced neuronal apoptosis due to
inhibiting tau protein phosphorylation.19 Edaravone is a free radical scavenger, and it has been used
in numerous patients with acute cerebral infraction in Japan from 2001.20 Previous study found that
edaravone was able to attenuate Aβ-induced oxidative stress and neurotoxicity.21 Recently, Wang’s
group found that edaravone had a potent capacity of inhibiting Aβ aggregation and attenuating
Aβ-induced oxidation in vitro. In animal models, they found that edaravone, given before or after the
oneset of Aβ deposition, alleviated oxidative stress, attenuated the downstream pathologies including
tau hyperphosphorylation, glical activation, neuroinflammation, neuronal loss, synaptic dysfunction,
and rescued the behavioral deficits of APPswe/PS1 mice.22 As mentioned above, these findings
suggested that DL-NBP and edaravone hold a promise as therapeutic agents for AD by targeting
multiple key pathways of the disease pathogenesis. As a further development of our work on the
design and synthesis of new multifunctional compounds with better pharmacological profiles,23-25
here we aimed to use a multi-target-directed drug design strategy to merge DL-BNP and edaravone
as hybrids for the treatment of AD. To explore the structure-activity relationships, the derivatives of
these hybrids (Scheme 1) were also synthesized.
In this letter, a series of DL-NBP-Edaravone hybrids were designed, synthesized and evaluated
for their biological activities, including inhibitory effects on Aβ aggregation, inhibition of MAO-A
and MAO-B, and antioxidant. To estimate the efficacy of these hybrids, the permeability of the
blood-brain barrier (BBB) was assayed by the parallel artificial membrane permeation assay for the
BBB (PAMPA-BBB) method.
The synthetic pathways of target compounds were summarized in Scheme 1. Phthalic anhydride
was reduced with NaBH4 in THF, and the reduction product was cyclized in 3N HCl at room
5

temperature to provide compound 2. Compound 2 was subjected to radical bromination, and
hydrolysis of the intermediate 3 gave 3-hydroxyisobenzofuran-1(3H)-one (4). The key intermediate 4
was first reacted with the appropriate Grignard reagents to give 5a-d in good yield, and subsequent
nitration of 5a-d by using potassium nitrate in sulfuric acid gave intermediates 6a-d. The nitro-group
of 6a-d was hydrogenated in the presence of Pd(OH)2/C (10%) to yield intermediates 7a-d.
Treatment of 7a-d with NaNO2 in conc. HCl gave the corresponding diazonium salt, which was
.
subsequently reduced by SnCl2 2H2O to give the substituted phenylhydrazines hydrochloride 8a-d.
The target compounds 9a-d were obtained from the reaction of 8a-d with ethyl acetoacetate in the
presence of sodium acetate in ethanol under reflux.26 While compounds 10a-d were obtained from
the cyclization of 8a-d with ethyl trifluoroacetoacetate in the presence of sodium acetate under reflux.
Due to the effect of trifluoromethyl, the cyclization reaction for the synthesis of 10a-d could not be
completely conducted under the same reaction conditions for that of 9a-d. In order to raise
cyclization reaction temperature, acetic acid was selected as solvent for the synthesis of 10-d. As
shown in Scheme 1, compounds 10a-d differed in tautomer conformation from 9a-d. The difference
in formation of 10a-d can be explained by the fact that, as a most electronegative atom, fluorine has
a very strong effect on the activities of nearby functional groups.27 Thus, in the case of 10a-d, due to
the strong electron withdrawing nature CF3 group, the methylene protons on C-4 may become highly
active resulting to enolize quickly. According to the literature,28 the tautomer stability of enol and
ketone can be identified and characterized by 1H and 13C NMR spectra (see Supporting Information).
1
In the H NMR spectra of target compouns, -OH proton signals can be found around δ 11.36-12.94
ppm, and signals of =CH- can be found around δ 5.92-6.09 ppm. C-4 signals of pyrazol can be found
δ 86.0 ppm in 13C NMR spectra. The results of NMR spectra indicated that compounds 10a-d existed
exclusively as enol form. In addition, the purity of 10a-d was confirmed by TLC and HPLC with
6

good purity, which was helpful to prove that 10a-d exsited in stable form of enol.
Scheme 1. Synthesis of DL-NBP-Edaravone hybrids 9a-d and 10a-d. Reagents and conditions: (i)
NaBH4, THF, at r.t. for 12h, then 3N HCl at r.t. for 12h; (ii) NBS, AIBN, benzene, at 75°C for 2.5h;
(iii) H2O, reflux for 3h; (iv) RMgBr, THF, at r.t. for 5h; (v) KNO3, H2SO4, at r.t. for 3h; (vi) H2,
Pd(OH)2, at r.t. for 24h; (vii) NaNO2, HCl, at 0-5°C for 1h, then SnCl2•2H2O, HCl, at 0-5°C for 2h;
(viii)ethyl acetoacetate, AcONa, EtOH, reflux for 5h; (ix) ethyl trifluoroacetoacetate, AcONa, AcOH,
reflux for 3h.
Aβ, especially Aβ1-42, and its aggregates have been considered as a primary etiological factor
triggering a neurotoxic cascade and finally causing neurodegeneration in AD brains.29 Thus, the
inhibition of self-induced Aβ1-42 aggregation of these hybrids was evaluated using a thioflavin-T
based fluorometric assay.23,30 Inhibitory activities as inhibition ratios at a test concentration of 25 μM
were displayed in Figure 1. All synthetic hybrids exhibited much higher inhibitory activity against
Aβ1-42 aggregation than the original lead compounds, DL-NBP (1.2%, at 25 μM) and edaravone
(20.1%, at 25 μM). However, compared with the reference compounds, curcumin (39.0%, at 25 μM),
compounds 9a-d and 10a showed more excellent inhibitory activities with inhibition ratios ranging
from 50.1% to 71.5%, while comounds 10c-d exhibited slightly lower inhibitory activities. Among
all the compounds, compound 9d exhibited the best anti-β-amyloid aggregation property (71.5%, at
25 μM). From the inhibition values (Figure 1), it was obvious that CF3-substituted hybrids 10b-d
7

exhibited lower inhibitory activities than the correspongding CH3-substituted compouns 9b-d. The
results revealed that trifluoromethyl group might not be favorable to their inhibitory activities against
self-induced Aβ1-42 aggregation. However, as shown in Figure 1, the length of alkyl side chains on
the phthalide ring had no significant effect on the inhibition of Aβ1-42 aggregation.
Figure 1. Inhibition of self-induced Aβ1-42 aggregation by compounds 9a-d, 10a-d, curcumin,
DL-NBP and edaravone. The thioflavin-T fluorescence method was used. All the compounds were
tested at 25μM.
In order to test their multipotent profile, compounds 9a-d and 10a-d have also been evaluated as
human MAO-A and MAO-B inhibitors following a fluorometric method as described in our previous
study,31 giving the IC50 values shown in Table 1, with clorgyline, rasagiline and iproniazid as
reference compounds. As shown in Table 1, all the tested compounds showed moderate, almost
equipotent inhibitory activities towards MAO-A and MAO-B with IC50 values in the range of
micromolar. To our satisfaction, all of them exhibited MAOs inhibitory activities much better than
both of the precursors (DL-NBP, edaravone). Compound 10b was the most potent MAO-A inhibitor
with an IC50 of 4.40 ± 0.18 μM, while the most potent MAO-B inhibitor was 9d with an IC50 of 4.36
± 0.24 μM. Similarly to the inhibition of Aβ1-42 aggregation, the length of alkyl side chains on the
phthalide ring exhibited no significant effect on the inhibition of MAOs. In general, these new
hybrids exhibited balance of inhibition for MAO-A and MAO-B and acted as non-selective MAO
inhibitors. It is known that non-selective MAO inhibitors are characterized by the risk of inducing
8

hypertensive crisis when dietary tyramine is ingested. Therefore, this non-selectivity profile may be a
deficiency for the discovery of novel, less toxic anti-Alzheimer drugs. However, the promising
inhibitory activities of these new hybrids against MAO-A and MAO-B laid the foundation for the
development of selective MAO inhibitors. And we would improve this deficiency in our follow-on
work.
Table 1. Inhibition of MAO activity, and oxygen radical absorbance capacity (ORAC, Trolox
equivalents) by DL-NBP-Edaravone hybrids and reference compounds
Compound
IC50 (μM) ± SDa
ORACe
Selectivity
Indexd
1.93
MAO-A
MAO-B
8.12 ± 0.68
12.86 ± 0.90
7.59 ± 0.31
15.65 ± 0.99
19.32 ± 0.81
15.20 ± 0.89
1.03 ± 0.03
0.62 ± 0.02
0.85 ± 0.02
9a
9b
9c
1.50
2.00
6.78 ± 0.42
7.24 ± 0.24
4.40 ± 0.18
4.36 ± 0.24
13.45 ± 1.06
21.50 ± 0.88
5.24 ± 0.48
1.56
0.54
0.20
1.21
1.40
NTf
1.18 ± 0.05
2.22 ± 0.07
2.37 ± 0.11
2.28 ± 0.14
2.21 ± 0.06
0.21 ± 0.01
9d
10a
10b
10c
10d
6.34 ± 0.45
8.57 ± 0.92
6.10 ± 0.26
18.20% ± 1.12%b
DL-NBP
11.20% ± 0.16%b
Edaravone
Clorgyline
Rasagiline
Iproniazid
n.a c
7.29% ± 0.38%b
NTf
0.00089
16.13
0.32
0.72 ± 0.03
NTf
0.0079 ± 0.0002
0.71 ± 0.03
1.37 ± 0.06
8.85 ± 0.34
0.044 ± 0.03
4.32 ± 0.31
NTf
NTf
a
IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50%
and are the mean of 3 independent experiments, each performed in triplicate (SD = standard
deviation).
b Percentages are the percent inhibition of MAO by tested compounds at 10 μM.
c
n.a. = no active. Compounds defined “no active” means a percent inhibition of less than 5.0% at a
concentration of 10 μM in the assay conditions.
d Selectivity index = IC50 (MAO-A)/IC50 (MAO-B).
e
The mean ± SD of the 3 independent experiments. Data are expressed as μM of Trolox
equivalent/μM of tested compound.
f NT = not tested.
9

As mentioned above, no clear structure-activtiy relationship (SAR) can be deduced from these
results. To structurely understand the interacting mode of these hybrids with MAO-A (PDB code:
2Z5X) and MAO-B (PDB code: 2V60), a molecular modeling study was performed using docking
program, AutoDock 4.2 package with Discovery Studio 2.0. On the basis of the in vitro inhibition
results, the best MAO-A inhibitor 10b and MAO-B inhibitor 9d were selected as typical ligands for
this assay. As shown in Figure 2A, the 3-(trifluoromethyl)-1H-pyrazol-5-ol moiety of 10b was close
to FAD cofactor, adopting hydrophobic interaction with Tyr444. And a hydrogen bond was formed
between hydroxy group and Asn181. Meanwhile, the alkyl side chain moiety interacted with many
hydrophobic amino acid residues, such as Leu97, Ala111, Ile3, Val210, Ser200 and Ile325. For
MAO-B, as shown in Figure2B, the edaravone moiety of 9d was located in the well-known binding
pocket of MAO-B, in close proximity to the FAD cofactor, and a π-π stacking interaction was formed
between the phenyl ring of edaravone and Tyr398. Another weak π-π stacking interaction was also
seen between phenyl ring of edaravone and Tyr326. More importantly, this complex was stabilized
by hydrogen bond interaction between 3-methyl-1H-pyrazol-5(4H)-one moiety of 9d and Tyr435.
The alkyl side chain moiety occupied the entrance cavity and interacted with lipophilic residues
Pro104, Phe103, Trp119, Ile199, Ile198, Phe168 and Leu167. Notably, two other hydrogen bonds
were observed between carbonyl oxygen and Tyr326 and Gln206. Overall, the docking results
rationalized the observed inhibition trends.
10

Figure 2. (A) Representation of compound 10b (colored by atom type) docked into the binding site
of hMAO-A (PDB code: 2Z5X); (B) Representation of compound 9d (colored by atom type) docked
into the binding site of hMAO-B (PDB code: 2V60). Hydrogen bonds are shown with the green
dotted lines. Pictures were generated with Discovery Studio 2.5. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
Oxidative stress is an important player of neuronal degeneration in AD. Its reduction is another
crucial aspect in designing multifunctional agents for AD treatment.32 In order to confirm that the
synthesized hybrids retain the antioxidant activity of edaravone, the potency of the target compounds
to eliminate free radicals was determined by measuring the oxygen radical absorbance capacity of
fluorescein (ORAC-FL),23,33 and the results were presented in Table 1. Their ability to scavenge
radicals was provided as a Trolox (a vitamin E analog) equivalent, with their relative potency at 1
μM compared with Trolox. DL-NBP and edaravone were also tested, with ORAC-FL values of 0.21
and 0.72 trolox equivalents, respectively. Particularly, and not surprisingly, all the target compunds
showed equal or better antioxidant activity than edaravone. Compounds 9a-d gave results with
0.62-1.18 trolox equivalents. Compounds 10a-d showed 2-fold higher antioxidative activity than
compounds 9a-d, with ORAC-FL values of 2.21-2.38 trolox equivalents. The results revealed that
the hydroxyl group produced by a keto-enol tautomerism is crucial for providing strong radical
scavenging ability. The putative free radical scavenging mechanism for these new hybrids was
similar to that for edaravone (Scheme 2).
Scheme 2. The proposed free radical scavenging mechanism of edaravone adopted from literature.34
The permeability through BBB of the anti-AD drug candidates should be assessed as early as
possible in the drug discovery process. A parallel artificial membrane permeability assay for BBB
11

(PAMPA-BBB) was used to determine the brain permeability of the synthesized compounds.31,35 We
used 11 commercial drugs as the reference compounds and we established the following ranges of
permeability: Pe (× 10-6 cm/s) > 3.44 for compounds with high BBB permeability; 3.44 > Pe (× 10-6
cm/s) > 1.61 for compounds with uncertain BBB permeability and Pe (× 10-6 cm/s) < 1.61 for
compounds with low BBB permeability. The representative compounds 9a, 9d, 10a and 10d were
selected for this assay. The results of the PAMPA-BBB assay (Table 2) indicates that compounds 9a
and 9d would be able to cross the BBB and reach their biological targets in the CNS. However,
compounds 10a and 10d were classified as low BBB permeation due to the increasing polarity
induced by the hydroxyl group.
Table 2. Permeability results Pe (× 10-6 cm/s) from the PAMPA-BBB assay for selected compounds
with their predicted penetration into the CNS.
Compd.a
Pe (× 10-6 cm/s)b
3.99 ± 0.16
4.25 ± 0.23
0.68 ± 0.02
0.73 ± 0.02
Prediction
CNS +
CNS +
CNS -
9a
9d
10a
CNS -
10d
a
Compounds were dissolved in DMSO at 5 mg/mL and diluted with PBS/EtOH (70:30). The final
concentration of each compound was 100 μg/mL.
b Data are the mean ± SD of three independent experiments.
In conclusion, a series of DL-NBP-Edaravone hybrids were designed, synthesized and evaluated
as dual inhibitors of amyloid-β aggregation and monoamine oxidases for the treatment of AD.
Compounds 9a-d displayed good inhibition of self-induced Aβ1-42 aggregation with inhibition ratio
57.7%-71.5%. However, the CF3-substituted hybrids 10c-d exhibited lower inhibitory activity than
the reference compound curcumin. For MAO, these new hybrids exhibited balance of inhibition for
MAO-A and MAO-B. And all of them exhibited MAOs inhibitory activities much better than both
the precursors (DL-NBP, edaravone). In addition, all the target compunds retained the antioxidant
activity of edaravone, showed equal or better antioxidant activity than edaravone. Furthermore,
compounds 9a and 9d were able to cross the blood-brain barrier. The promising results in all assays
12

demonstrated that the strategy behind the designing of compounds was rational and favourable.
Overall, these preliminary findings suggested that these new hybrids could be further investigated for
pharmacological development in AD therapy.
Acknowledgments
This work was supported in part by the Chinese National Natural Science Foundation (20872099),
the Research Fund for the Doctoral Program of Higher Education (20110181110079) and the
National Science and Technology Major Project on “Key New Drug Creation and Manufacturing
Program”(2013ZX09301304-002).
Supplementary data
Supplementary data (the synthesis and characterization of the new compounds as well as the detailed
description of the biological evaluation assays) associated with this article can be found, in the online
version.
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(9d). To a mixture of intermediate 8d (80 mg, 0.268 mmol) and sodium acetate (22 mg, 0.268
mmol) in ethanol was added ethyl acetoacetate (0.036 mL, 0.281 mmol). The reaction mixture
was heated to reflux for 5 h under an argon atmosphere. After complete reaction, the solvent was
evaporated under reduced pressure. Then water (20 mL) was added to residue and the mixture
was extracted with ethyl acetate (15 mL×3). The combined organic layers were washed with
brine (15 mL×2), dried over sodium sulfate, and concentrated in vacuo. The residue was purified
by preparative thin-layer chromatography (developing with petroleum ether/ethyl acetate=2:1) to
give 9d as yellow oils. 44.0% yield; 1H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H, Ar-H), 8.31 (dd,
1H, J1 = 8.0 Hz, J2 = 2.0 Hz, Ar-H), 7.43 (d, 1H, J = 8.0 Hz, Ar-H), 5.47 (dd, 1H, J1 = 7.6 Hz, J2 =
4.0 Hz, CH), 3.49 (s, 2H, CH2) 2.23 (s, 3H, CH3), 2.06-1.99 (m, 1H, 1/2 × CH2), 1.79-1.73 (m,
1H, 1/2 × CH2), 1.47-1.42 (m, 2H, CH2), 1.40-1.26 (m, 8H, 4 × CH2), 0.88 (t, 3H, J = 7.2 Hz,
CH3); 13C NMR (100 MHz, CDCl3) δ 170.6, 170.2, 157.0, 145.7, 139.0, 126.7, 124.0, 122.0,
115.0, 81.3, 43.0, 34.7, 31.6, 29.2, 28.9, 24.6, 22.5, 16.9, 14.0; HRMS (ESI) calcd for
C19H25N2O3 [M+H] + m/z: 329.1865, found 329.1862. Purity: 99.0% (by HPLC).
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