Design, synthesis and evaluation of 3-arylidene azetidin-2-ones as potential antifungal agents against Alternaria solani Sorauer

Article abstract of DOI:10.1016/j.bmc.2017.11.003

A new concise and facile method was explored to synthesize a collection of new 3-arylidene azetidin-2-ones, which could be regarded as the derivatives of the hybrid scaffold of bioactive natural cinnamamide and heterocycle azetidi-2-one. The structures of

Full text of DOI:10.1016/j.bmc.2017.11.003

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
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Bioorganic & Medicinal Chemistry
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Design, synthesis and evaluation of 3-arylidene azetidin-2-ones as
potential antifungal agents against Alternaria solani Sorauer
a
Wang Delong a, Wu Yongling a, Wang Lanying a,b, Feng Juntao a, , Zhang Xing
⇑
a Research & Development Center of Biorational Pesticide, Shaanxi Research Center of Biopesticide Engineering & Technology, Key Laboratory of Plant Protection Resources and
Pest Management of Ministry of Education, Northwest A&F University, Yangling 712100, China
b College of Environment and Plant Protection, Hainan University, Haikou, Hainan 570228, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new concise and facile method was explored to synthesize a collection of new 3-arylidene azetidin-2-
ones, which could be regarded as the derivatives of the hybrid scaffold of bioactive natural cinnamamide
and heterocycle azetidi-2-one. The structures of the synthesized compounds were characterized by 1H,
13C NMR, and MS; and their antifungal activity were evaluated against Alternaria solani Sorauer. These
antifungal data were subjected to a quantitative structure–activity relationship (QSAR) analysis using
Codessa software on the basis of the results from B3LYP/6-31G(d,p) quantum calculations. The best
regressive model revealed that potentially more active compounds should have low dipole moments
and QC-min (minimal net atomic charge for a C atom), and high QO-max (maximal net atomic charge for
an O atom) and QN-min (minimal net atomic charge for an N atom). The most potent compound 7k could
lead to intracellular accumulation of reactive oxygen species, dissipation of mitochondrial transmem-
brane potential, and an autophagy-like cell death process in A. solani Sorauer. Taken together, these
results laid the foundation for further design of improved crop-protection agents based on this hybrid
scaffold.
Received 21 August 2017
Revised 26 October 2017
Accepted 2 November 2017
Available online xxxx
Keywords:
3-Arylidene azetidin-2-ones
Antifungal activity
QSAR model
Subcellular effect
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
chances for the development of resistance.9 Accordingly, there is
a wake-up call currently arousing a regenerated interest in discov-
Plant diseases have greatly impacted human civilizations since
the dawn of agriculture. These impacts, however, have not abated
yet. In fact, the emerging infectious diseases of plants caused by
fungi and fungal-like oomycetes have been recognized as a sharply
rising worldwide threat to food security and biodiversity conserva-
tion1–4; and this threat is increasingly aggravated, not only due to
the coevolved phytopathogens following their hosts to new
ecosystems, but as a result of introduced crops encountering new
pathogens.5 What is worse, global trade and transportation,
together with climate changes, will exaggerate this situation.1,2
Fortunately, since 1960s, a vast evolving array of pesticides pro-
vided by agrochemical industry have played a central role in
crop-disease management strategies and, foreseeably, it is unlikely
to change in the future.6–8 However, for nearly three-quarters of a
century, many fungicide products have vanished from the market,
because their ecotoxicological and environmental features no
longer satisfy the growingly stringent regulations.9 Survivals of
products from these challenges will still disappear, owing to the
ery of new antifungal compounds, especially for these with new
target sites (or mode of actions, MoAs).
Historically, phytochemicals and botanical-derived scaffolds
have innovatively been the invaluable inspirational resources for
discovery and development of new pesticides.10–15 Botanical
metabolites are the miraculous masterworks from nature’s work-
shop. They innately break down readily and often their adverse
effects are not as long-lasting as those of synthetic pesticides,
thereby giving less heavy burden on the environment and less pro-
found consequences on non-target organism. A strikingly diverse
array of constitutive and inducible phytochemicals take various
MoAs to protect plants from their enemies,16 which may indeed
be the result of millions of years’ coevolution of plants with their
predators. That is the reason why many botanical phytochemicals
have long been used as pesticides and have extensively served as
the templates for a great many of commercial synthetic pesticides
that are on the market today.12,13,15
Cinnamamide and its natural derivatives occur widely in many
higher plants17 and they showed diverse beneficial biological activ-
ities, such as antioxidant,18 antimicrobial,19 larvicidal,20,21 nemati-
cidal, 21antiinflammatory22 and animal repellent23,24 activities. On
⇑
Corresponding author.
E-mail address: fengjt@nwafu.edu.cn (F. Juntao).
https://doi.org/10.1016/j.bmc.2017.11.003
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Delong W., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.003

2
W. Delong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
the other hand, ring systems, particular heterocycles, are funda-
mental cornerstones for building of most of drugs on the market
today.25 This is mainly because they are major determinants of
molecular properties, such as the electronic distribution and scaf-
fold rigidity, and key contributors to modulate pharmacokinetic
properties of molecules, such as molecular reactivity, metabolic
stability.26 Of these rings, azetidin-2-one, a vital pharmacophore,
plays a significant role in a wide range of bioactive molecules,
including penicillins, nocardicins, clavulanic acid and sulbactams,
which have been widely used to treat bacterial infections and
microbial diseases.27 In addition, more recent renewed attention
has focused on this versatile nucleus to synthesize compounds
with diverse biological activities like human tryptase28 and chy-
mase inhibitory activity,29 antiinflammatory,30 antitumor31 and
antifungal activity.32–34
In present study, we explored a concise and facile approach to
synthesize a collection of new 3-arylidene azetidin-2-ones, which
could be regarded as the derivatives of the hybrid scaffold of cinna-
mamide (or its derivatives) and azetidin-2-one. Their antifungal
activity was screened and a quantitative structure-activity rela-
tionship (QSAR) model was established via Codessa software.
Besides, the MoA of the most potent compound on the test fungus
was preliminarily investigated using transmission electron micro-
scope (TEM) and fluorography techniques. These results may lay
the foundation for the exploitation of this hybrid scaffold to seek
potential crop-protection agents.
involved in a SN20 attack on allyl bromides to give kinetic prod-
ucts,37 SN2 products 4 were predominantly formed in polar solvent
acetonitrile with moderate to good yields (41–71%) in our most
cases.
With SN2 products 4a–p in hand, our following attention
focused on their viable access to target compounds 5a–p. Although
some of 4 have already been proved as the precursors for synthesis
of cyclized products via a two-step process (Scheme 2), we would
like to explore a facile single-step procedure for the cyclization
reaction. Conventional bases such as ethyl magnesium bromide
(EtMgBr), n-butyllithium (n-BuLi), potassium tert-butoxide (t-
BuOK) and lithium hydroxide (LiOH) were unable to implement
this cyclization reaction (Table 1, entries 1–4). Nevertheless, bis
[bis(trimethylsilyl)amino]tin(II) (Sn[N(TMS)2]2) was found to be a
good reagent in the cyclization of SN20 product to
a-methylene-
b-lactams.37 Inspired by this previously reported method, we
attempted to perform this reaction by using a cheap and easily
available base lithium bis(trimethylsilyl)amide (LHMDS) instead
of Sn[N(TMS)2]2. Much to our delight, by means of the optimized
condition (Table 1, entries 5–7), the reaction afforded the desired
5a–p in moderate to good yields (43–74%) when SN2 products
4a–p were treated with 2.0 equivalents of LHMDS in THF at ꢀ20
°C. By comparison with the 1H NMR spectra of 4, it was conve-
niently possible to determine the reaction from the 1H NMR spec-
tra of 5 where the methoxy (–OCH3) signals (dH around 3.80 ppm)
disappeared, the methylene (–CH2–) signals showed a striking
downfield shift from ꢁ3.60 ppm to ꢁ4.10 ppm due to the deshield-
ing effect of amide group, and the olefinic proton signals (–CH@C)
were shifted dramatically upfield from ꢁ7.80 ppm to ꢁ6.90 ppm
owing to the olefinic protons spatially far away from the deshield-
ing center of carbonyl group in the cyclic conformation (Supple-
mentary Materials, Fig. S1).
2. Results and discussion
2.1. Synthesis methods
The synthetic route begins with Baylis-Hillman reaction of alde-
hydes 1 with methyl acrylate in the presence of 1,4-diazabicyclo
[2.2.2]octane (DABCO) (Scheme 1). Generally, these reactions were
run neat at room temperature except the insoluble substrates 4-
nitrobenzaldehyde (1o) and 2-naphthaldehyde (1p) whose reac-
tions were carried out in THF respectively. The Baylis-Hillman
adducts (2) were next converted into allyl bromides intermediates
(3) by treatment with concentrated HBr (47%, w/w) and H2SO4
(98%, w/w), a transformation that undergoes allylic rearrangement
and provides only the thermodynamically favored (Z)-configurated
olefin.35,36 Then the thermodynamically controlled introduction of
N-nucleophiles was performed by the SN2 replacement of the
allylic bromides with amines to afford 4.36 To optimize the sub-
stituents (R) on the benzene ring, we selected the simple liquid
n-propylamine at room temperature as the N-nucleophile and kept
it fixed before the following optimization of N-substituents.
Although it has been reported that the N-nucleophiles could be
With the optimized substituent 4-Cl being fixed on the benzene
ring, finally, we investigated the effect of N-substituents on the
antifungal activity. Similarly, the precursors 6a–m were generated
in moderate to good yields (37–69%) by reaction of 3c with differ-
ent N-nucleophiles (Scheme 3). The following cyclization reactions
proceeded smoothly and efficiently except that the tert-butyl pre-
cursor 6d didn’t react at all. All the NMR spectra of new com-
pounds were included in Supplementary Materials.
2.2. Antifungal activity and QSAR study of the synthesized compounds
The lead compound 5a was primarily selected to test its antifun-
gal activity against eight economically important phytopathogenic
fungi Fusarium graminearum Sehw., Gaeumannomyces graminis var.
graminis, Pyricularia oryzae Cav., Sclerotinia sclerotiorum (Lib.) de
Bary, Valsa mali Miyabe et Yamada, Colletotrichum orbiculare,
O
OH
O
O
O
DABCO
CHO
HBr/H2SO4
DCM, 0 oC to rt,
overnight
O
O
R
R
R
neat or THF, rt, 7d
Br
1a-p
2a-p
3a-p
O
O
1
3
NH2
O
Conditions
R
R
N
4
K2CO3, CH3CN,
rt, 1h
NH
5a-p
4a-p
Scheme 1. The synthetic routine to the target compounds 5a–p.
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W. Delong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Table 2
O
The mycelial growth inhibition rates of compound 5a against eight phytopathogenic
fungi at 50.0 g/mL.
1. NaOH,
+
-
OH
l
MsCl, Bu4N HSO4
KHCO3
EtOH/H2O
+
Mycelial growth inhibition rate (%)a
2. HCl/H2O
Fungi species
NH2
the reported method
present study
•
Cl-
CHCl3/H2O
F. graminearum Sehw.
G. graminis var. graminis
P. oryzae Cav.
S. sclerotiorum (Lib.) de Bary
V. mali Miyabe et Yamada
C. orbiculare
52.1 1.6
39.6 1.6
16.7 2.2
23.4 1.7
14.5 2.6
38.4 2.8
11.1 1.5
67.9 2.4
O
O
O
R
R
N
NH
B. cinerea
A. solani Sorauer
5a
5o
R= 4-NO2
R= H;
a
Data are expressed as the means SD (n = 3).
4a
4o
R= 4-NO2
R= H;
Scheme 2. The previously reported method for synthesis of compound 5a and 5o.
by Codessa including constitutional, topological, geometrical, elec-
trostatic, quantum chemical, and thermodynamic ones. The opti-
mal number of molecular descriptors within the model were
determined by the ‘‘breaking rule” using the heuristic method in
Codessa.39 According to this rule, the squared correlation coeffi-
cients (R2) were plotted versus the number of descriptors and the
regression calculation would be halted at the ‘‘breaking point”
Table 1
Results of different conditions for cyclization of b-aminoester 4a to
lactam 5a.
a-arylidene-b-
Entry
Reagents/conditions
Product yield
1
2
3
4
5
6
7
EtMgBr (1.5 equiv), THF, ꢀ20 °C to r.t., 2 h
n-BuLi (1.5 equiv), THF, ꢀ20 °C to r.t., 2 h
t-BuOK (1.5 equiv), THF, ꢀ20 °C to r.t., 2 h
LiOH (2.0 equiv), THF, 0 °C to relux, 1 h
LHMDS (1.0 equiv), THF, 0 °C to r.t., 2 h
LHMDS (1.5 equiv), THF, ꢀ20 °C, 1 h
LHMDS (2.0 equiv), THF, ꢀ20 °C, 1 h
No reaction
No reaction
Mess
where the statistical increments of R2 ( R2) become less significant
D
(<0.02). As illustrated in Fig. 1, the breaking point occurred at four
descriptors, and the values of the four descriptors and the four-
parameter model are given in Tables 3 and 4 respectively.
The statistical parameters of the best QSAR model are as fol-
Mess
26%a,b
57%a,b
74%a,b
lows: R2 = 0.8778, F = 41.31, s2 = 0.0027. In Table 4, X and
DX are
a
On TLC plate only one product spot was visualized by UV light and Dragendoff’s
reagent.
the regression coefficients of the QSAR equation and their standard
error, respectively. The descriptors in the model are given in
descending order of significance, according to their statistical t-test
value. The leave-one-out (LOO) cross-validation approach was
used to validate the obtained model. The LOO cross-validated
QCV2 was 0.9008 and it did not differ significantly from the R2, indi-
cating that the predictive power for this model was retained after
b
isolated yield.
Botrytis cinerea, Alternaria solani Sorauer. The results were summa-
rized in Table 2. As shown therein, compound 5a was active against
those eight fungi to various extent at the concentration of 50.0 lg/
mL and exhibited the highest inhibitory activity against A. solani
Sorauer with mycelial growth inhibition rate of 67.9%. Hence, A.
solani Sorauer was selected as the target fungi in the following lead
optimization. Keeping N-n-propyl substitution fixed, we first inves-
tigated the influence of variations of substituents at benzene ring
on the antifungal activity. As a result, the 4-Cl substitution dis-
2
2
removing any one datum. The external Rext and Qext values were
0.8963 and 0.8931 respectively (Table S1, Supplementary Materi-
als), which were similar to the training set (R2), thus simultane-
ously demonstrating the robustness of the generated model.
From this best QSAR model,
the antifungal activity. The dipole moment
cally significant descriptor. The is responsible for the interactions
l, QC-min, QO-max and QN-min govern
l
is the most statisti-
l
played the most potent activity with EC50 of 98.2 lM (Table 3)
of intermolecular dipole-dipole and dipole-induced dipole, which
and, accordingly, we subsequently optimized the N-substitutions
plays an important role in the interactions of drug with recep-
with 4-Cl atom at benzene ring. Consequently, among these deriva-
tor.40,41 The negative value of coefficient for
l
indicated that mole-
tives, compound 7k had the highest potency with EC50 of 37.6
which was comparable to the commercial procymidone (30.6
(Table 3).
l
M,
cules with lower
l would possess higher antifungal activity (e.g.
lM)
compound 7i–k). For our synthesized molecules, their polarities
have a positive correlation with their total net dipole moments.
Thus, it is supposed that the binding sites of these compounds
might be located at a relative nonpolar pocket.
Generally, a molecule could be considered as a group of nuclei
bound together by overlapped electron orbitals. This structural
In order to probe the main molecular factors that contribute to
the antifungal activity, we conducted follow-up QSAR analyses.
The most DFT method together with B3LYP hybrid functional
was employed to obtain the lowest energy confirmations of the
molecules since it has been demonstrated that this method could
improve the accuracy of the results and give more reliable QSAR
models, in comparison with the traditional semi-empirical
method.38 After conversion of the Gaussian output files into
Codessa software compatible formats (Ampac output files), hun-
dreds of molecular descriptors of each molecule were calculated
illustration leads to deriving descriptors such as
l and atomic
charge. The other three descriptors are related to atomic charges.
Electrical charges in the molecules conduce to the molecular elec-
trostatic interactions with their binding sites.40,42 Indeed, atomic
charges or local electron densities has been widely used to inter-
O
O
O
LHMDS
O
R'NH2
O
THF, -20 oC, 1h
N
R'
K2CO3, CH3CN,
r.t., 1~3h
Cl
Br
Cl
NH
R'
Cl
3c
6a-m
7a-c,e-m
Scheme 3. The synthetic routine to the target compounds 7a–c, e–m.
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4
W. Delong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 3
Antifungal activity of compounds 5a–p and 7a–c, e–m against A. solani Sorauer and descriptors of the title compounds.
a
b
Compd.
R
R0
l
(Debye)
QC-min (a.u.)
QO-max (a.u.)
QN-min (a.u.)
EC50
(lM)
pEC50
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
7a
7b
7c
7e
7f
7g
7h
7i
7j
7k
7l
7m
H
4-F
4-Cl
4-Br
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3.7620
2.5420
2.7240
2.6010
4.0910
4.0590
4.0860
4.1970
4.7310
4.9460
2.6650
4.0290
4.3390
3.6040
3.6020
3.8370
2.6440
2.7270
2.6550
2.6710
2.5430
2.5990
2.5310
2.7360
3.8730
3.7130
3.9170
2.6110
–
ꢀ0.2085
ꢀ0.2087
ꢀ0.2087
ꢀ0.2088
ꢀ0.2085
ꢀ0.2085
ꢀ0.2085
ꢀ0.2085
ꢀ0.2114
ꢀ0.2186
ꢀ0.2087
ꢀ0.2085
ꢀ0.2086
ꢀ0.2085
ꢀ0.2785
ꢀ0.2085
ꢀ0.2180
ꢀ0.2111
ꢀ0.2111
ꢀ0.2184
ꢀ0.2106
ꢀ0.1953
ꢀ0.2105
ꢀ0.1895
ꢀ0.1909
ꢀ0.1913
ꢀ0.2063
ꢀ0.1891
–
ꢀ0.2894
ꢀ0.2872
ꢀ0.2867
ꢀ0.2859
ꢀ0.2901
ꢀ0.2900
ꢀ0.2902
ꢀ0.2904
ꢀ0.2907
ꢀ0.2910
ꢀ0.2869
ꢀ0.2898
ꢀ0.2843
ꢀ0.2902
ꢀ0.2785
ꢀ0.2895
ꢀ0.2889
ꢀ0.2866
ꢀ0.2899
ꢀ0.2895
ꢀ0.2867
ꢀ0.2895
ꢀ0.2873
ꢀ0.2929
ꢀ0.2910
ꢀ0.2893
ꢀ0.2873
ꢀ0.2937
–
ꢀ0.3028
ꢀ0.3018
ꢀ0.3016
ꢀ0.3020
ꢀ0.3025
ꢀ0.3024
ꢀ0.3026
ꢀ0.3027
ꢀ0.3022
ꢀ0.3022
ꢀ0.3016
ꢀ0.3030
ꢀ0.3027
ꢀ0.3037
ꢀ0.3001
ꢀ0.3025
ꢀ0.2989
ꢀ0.3014
ꢀ0.3091
ꢀ0.3003
ꢀ0.2988
ꢀ0.2983
ꢀ0.2998
ꢀ0.3117
ꢀ0.3126
ꢀ0.3130
ꢀ0.3135
ꢀ0.3106
–
283.4 18.4
172.5 6.8
98.2 14.9
192.0 16.4
107.7 12.6
402.2 12.7
161.7 8.6
312.9 18.7
264.9 13.9
450.4 29.0
147.6 13.2
195.1 16.7
306.4 26.4
200.9 21.4
388.4 28.0
583.2 35.9
179.5 22.1
147.2 7.2
211.6 12.4
282.0 16.5
117.2 9.7
495.3 31.1
399.3 28.2
80.4 5.6
3.5476
3.7632
4.0079
3.7167
3.9678
3.3956
3.7913
3.5046
3.5769
3.3464
3.8309
3.7097
3.5137
3.6970
3.4107
3.2342
3.7459
3.8321
3.6745
3.5498
3.9311
3.3051
3.3987
4.0947
4.2168
4.4248
3.7066
3.6008
–
4-CH3
4-CH3CH2
4-(CH3)2CH
4-(CH3)3C
4-CH3O
4-CH3CH2O
3-Cl
3-CH3
2-Cl
2-CH3
4-NO2
3,4-Benzo–
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
(CH3)2CH
CH3(CH2)2CH2
(CH3)2CHCH2
CH3CH2(CH3)CH
CH3(CH2)4CH2
(CH2)5CH
CH3(CH2)6CH2
C6H5
4-FC6H5
4-ClC6H5
4-BrC6H5
4-CH3C6H5
–
60.7 6.6
37.6 3.6
196.5 13.0
250.7 24.4
30.6 4.6
4-Cl
4-Cl
4-Cl
–
Procymidonec
Note:
l, total dipole of the molecule; QC-min, minimal net atomic charge for a C atom; QO-max, maximal net atomic charge for an O atom; QN-min, minimal net atomic charge for
an N atom.
a
All 50% maximal effective concentration (EC50) values are presented as the means SD (n = 3), lM.
Values were obtained from pEC50 (M).
Commercial procymidone was used as control.
b
c
positive, implying their positive correlations with activity. How-
ever, QN-min has a detrimental effect on activity due to its negative
value of coefficient. The net atomic charges of compounds 5c and
7i–k are shown in Fig. 2. Taken together, these quantitative analy-
ses give some useful hints for designing potential antifungal agents
in the future.
1.0
0.8
0.6
0.4
0.2
2.3. Effect of compound 7k on subcellular structures in A. solani
Sorauer
The effect of the most potent compound 7k on subcellular
structures in A. solani Sorauer was observed by ultrastructural
imaging using TEM. In the control (Fig. 3A), the cytoplasmic mem-
brane was visible and exhibited good adhesion to the outer cell
wall, which was a bright fibrous layer. Regularly shaped mitochon-
dria with membranous cristae displayed normal inner and outer
membranes. Normal sized vacuoles with low density contents
were loosely distributed in cells. The control fungal cells had dense
cytoplasm due to the presence of proteic and lipidic materials,
indicating their normal functionality.
1
2
3
4
5
6
7
8
Number of descriptors
Fig. 1. R2 values versus the number of the descriptors used for the model.
pret the chemical reactivity and physicochemical properties of
molecules.40,42 The values of coefficient for QC-min and QO-max are
Table 4
The best four descriptor QSAR model with R2 = 0.8778, F = 41.31, s2 = 0.0027.
Descriptor No.
X
D
X
t-Test
Descriptora
0
1
2
3
4
4.7348e+00
ꢀ1.7493eꢀ01
7.8452e+00
1.6488e+00
ꢀ4.9969e+00
1.2322e+00
1.4449eꢀ02
1.7681e+00
5.6585eꢀ01
3.1124e+00
3.8426
Intercept
l
QC-min
QO-max
QN-min
ꢀ12.1067
4.4371
2.9138
ꢀ1.6055
a
Descriptors are defined in Table 3.
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W. Delong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Fig. 2. The net charges of atoms C, N and O for compounds 5c, 7i-k.
Fig. 3. Effect of compound 7k on subcellular structures of A. solani Sorauer. A, control; B, 12 h; C, 24 h; D, 48 h. (CW) cell wall, (M) mitochondria; (V) vacuole, (PM) plasmic
membrane, (AV) autophagy-like vesicle, (black arrow) autophagy-like process.
After treatment with compound 7k for 12 h, the fungal cells
already showed some alternations of the cytoplasm membranes
that became irregular in thickness and detached from cell wall to
some extent (Fig. 3B). Particularly, the number of mitochondria
increased and many of them were hypertrophic. After 24 h, many
cells were damaged, showing unclear cytoplasm membranes, pres-
ence of autophagy-like vesicles, and reduced density of cytoplas-
mic matrix (Fig. 3C). When the treatment up to 48 h, the
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6
W. Delong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
cytoplasm was clearly disrupted and looked empty (Fig. 3D), as a
result of the autophagy-like process and the cell’s contents leakage
without the prevention of cell membranes. There were no orga-
nelles discernible due to the high vacuolization.
Considering the above symptom observations, it could therefore
be concluded that the mitochondria were one of the primary sub-
cellular targets of compound 7k. As is known, mitochondria are the
primary intracellular site generating ATP via oxidative phosphory-
lation and the major source of reactive oxygen species (ROS) gen-
eration from mitochondrial respiratory chain.43,44 Thus, the redox
homeostasis in mitochondria is pivotal for organism, because if
this balance is broken and the accumulation of ROS is not limited,
it diffuses and impairs mitochondrial DNA and proteins and other
cellular compartments.45,46 Meanwhile, autophagy, one type of
programmed cell death, is a major catabolic pathway in eukaryotic
cells, which mainly responds to both extracellular stress (starva-
tion) and intracellular stress (accumulation of damaged organelles
or proteins). As has been verified previously, the autophagy could
be triggered by mitochondrial dysfunction.47–50
In our case, toxic symptoms of A. solani Sorauer showed aber-
rant hypertrophy in mitochondria after treatment with compound
7k for 12 h, indicating mitochondrial dysfunction occurred. There-
fore, we corroborated this fact using a mitochondrial transmem-
brane potential-sensitive dye JC-1. The healthy cells have high
Fig. 5. Visualization of oxidative stress symptoms in A. solani Sorauer after
treatment with compound 7k. ROS within hyphae were detected using the
fluorescent dye DCFH-DA. Green signals correspond to the ROS accumulation in
cells.
mitochondrial membrane potential (DWm) and JC-1 selectively
incorporates into mitochondria to spontaneously form aggregates
at this high potential, exhibiting red fluorescence. In the damaged
and increase in ROS accumulation.51–54 Taken together, these
results laid the foundation for the design of new mitochondria-tar-
geted fungicides based on the scaffold 3-arylidene azetidin-2-one.
cells, the
monomeric form, which produces green fluorescence. As shown in
Fig. 4, significant dissipation of Wm was well observed. The mean
DWm becomes low and this dissipation keeps JC-1 in the
D
green/red fluorescence ratio of the dye increased from 0.013
0.003 in the control to 0.921 0.085 after treatment. Additionally,
we examined the ROS level changes in the treated fungus using
DCFH-DA probe which is non-fluorescent but becomes the highly
fluorescent form (20,70-dichlorofluorescein) once oxidized by ROS.
The results (Fig. 5) demonstrated that compound 7k could induce
ROS accumulation. As mentioned above, this ROS accumulation
mighty be the plausible reason for mitochondria damages and con-
sequently contributed to the autophagy-like process and other cell
impairments. Besides here, many mitochondria-targeted drugs
have been reportedly demonstrated to alter the mitochondria-
3. Conclusion
We have developed a new concise and facile method to synthe-
size a collection of new 3-arylidene azetidin-2-ones. Their struc-
tures were characterized and their antifungal activity were
evaluated against Alternaria solani Sorauer. The established QSAR
model indicated that potentially more active compounds should
have low dipole moment and QC-min, and high QO-max and QN-min
.
The most potent compound 7k could lead to intracellular accumu-
lation of reactive oxygen species, dissipation of mitochondrial
transmembrane potential, and an autophagy-like cell death pro-
cess in A. solani Sorauer. Taken together, these results laid the foun-
related cell status, including reduction in
DWm and ATP production
Fig. 4. Effect of compound 7k on the mitochondrial transmembrane potential of A. solani Sorauer. The mitochondrial transmembrane potential was assessed using the
fluorescent potentiometric dye JC-1. Red signals show cells containing mitochondria with high transmembrane potential and green signals represent mitochondria with low
potential.
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W. Delong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
dation for further design of improved crop-protection agents based
on the scaffold 3-arylidene azetidin-2-one.
4.2.4. (Z)-Methyl 2-(bromomethyl)-3-(4-bromophenyl)acrylate (3d)
Yield 77%; yellow oil; 1H NMR (500 MHz, CDCl3): d 7.73(s, 1H),
7.52(dd, J = 8.5, 2.0 Hz, 2H), 7.44(dd, J = 8.4, 1.1 Hz, 2H), 4.34(s, 2H),
3.88(s, 3H); 13C NMR (125 MHz, CDCl3): d 166.4, 141.6, 133.1,
132.2, 131.1, 129.3, 124.1, 52.6, 26.3.
4. Experimental
4.1. General
4.2.5. (Z)-Methyl 2-(bromomethyl)-3-(p-tolyl)acrylate (3e)
Yield 61%; yellow oil; 1H NMR (500 MHz, CDCl3): d 7.82(s, 1H),
7.50(d, J = 7.8 Hz, 2H), 7.28(d, J = 7.6 Hz, 2H), 4.43(s, 2H), 3.89(s,
3H), 2.41(s, 3H); 13C NMR (125 MHz, CDCl3): d 166.8, 143.1,
140.1, 131.4, 129.9, 129.6, 127.8, 52.4, 27.1, 21.5.
All reagents were commercially purchased from Shanghai
Macklin Biochemical Co., Ltd. (Shanghai, China) and used without
further purification. All solvents were analytical grade and directly
used, unless otherwise noted. Tetrahydrofuran (THF) was distilled
from sodium and benzophenone under nitrogen atmosphere
before use. Dry acetonitrile (CH3CN) was distilled from calcium
hydride and stored in 4 Å molecular sieves. The air-free and mois-
ture-free reactions were carried out under an inert nitrogen atmo-
sphere using the standard Schlenk techniques. Thin layer
chromatography (TLC) was performed on glass plates coated with
GF254 silica gel to monitor the reaction progress. Spots were visu-
alized by UV light, ninhydrin reagent (for amines), or Dragendoff’s
reagent (for amides). Column chromatography was performed
using silica gel (200–300 mesh). 1H and 13C NMR spectra were
recorded at room temperature on a Bruker AMX500 (1H at 500
MHz, 13C at 125 MHz) magnetic resonance spectrometer. 1H and
13C chemical shifts were reported in ppm and they were calibrated
according to the CDCl3 peaks at 7.27 ppm and 77.2 ppm, respec-
tively. ESI-MS spectra were recorded with a AB Sciex API 2000
LC/MS system. Melting points (m.p.) were determined on a X-4
melting point apparatus (uncorrected).
4.2.6. (Z)-Methyl 2-(bromomethyl)-3-(4-ethylphenyl)acrylate (3f)
Yield 65%; yellow oil; 1H NMR (500 MHz, CDCl3): d 7.82(s, 1H),
7.58(d, J = 7.8 Hz, 2H), 7.30(d, J = 8.1 Hz, 2H), 4.43(s, 2H), 3.88(s,
3H), 2.70(q, J = 7.6 Hz, 2H), 1.27(t, J = 7.6 Hz, 3H); 13C NMR (125
MHz, CDCl3): d 166.8, 146.4, 143.2, 131.6, 130.0, 128.5, 127.7,
52.4, 28.8, 27.2, 15.3.
4.2.7. (Z)-Methyl 2-(bromomethyl)-3-(4-isopropylphenyl)acrylate
(3g)
Yield 71%; yellow oil; 1H NMR (500 MHz, CDCl3): d 7.86(s, 1H),
7.58(d, J = 8.2 Hz, 2H), 7.37(d, J = 8.2 Hz, 2H), 4.47(s, 2H), 3.92(s,
3H), 2.95–3.03(m, 1H), 1.32(t, J = 6.9 Hz, 6H); 13C NMR (125 MHz,
CDCl3): d 166.8, 151.0, 143.1, 131.8, 130.0, 127.7, 127.0, 52.4,
34.1, 27.2, 23.7.
4.2.8. (Z)-Methyl 2-(bromomethyl)-3-(4-(tert-butyl)phenyl)acrylate
(3h)
Yield 55%; yellow oil; 1H NMR (500 MHz, CDCl3): d 7.82(s, 1H),
7.55(d, J = 8.4 Hz, 2H), 7.49(d, J = 8.4 Hz, 2H), 4.44(s, 2H), 3.88(s,
3H), 1.32(s, 9H); 13C NMR (125 MHz, CDCl3): d 166.8, 153.2,
143.0, 131.4, 129.8, 127.7, 125.9, 52.4, 34.9, 31.1, 27.2.
4.2. General procedure for the preparation of cinnamyl bromide
intermediates 3a–p55
The Baylis-Hillman adducts 2a–p were synthesized by coupling
of aromatic aldehydes to methyl acrylate in the presence of DABCO
without solvent according to the previously reported method. Par-
ticularly, the reaction systems of 4-nitrobenzaldehyde and 2-naph-
thaldehyde were carried out in THF. To prepare the cinnamyl
bromide intermediates, a solution of corresponding Baylis-Hillman
adduct (30.0 mmol) in DCM (2.0 mol/L) was cooled to 0 °C. To the
above stirring solution was added conc. HBr (48%, 9.5 mL) and then
conc. H2SO4 (98%, 8.6 mL). The resulting mixture was stirred over-
night at r.t. and then 30 mL H2O and 20 mL DCM were carefully
added under ice bath. The organic phase was separated, washed
with H2O (30 mL ꢂ 3), dried over Na2SO4, and concentrated. The
desired product was obtained through column chromatography
with petroleum ether/EtOAc (20/1).
4.2.9. (Z)-Methyl 2-(bromomethyl)-3-(4-methoxyphenyl)acrylate (3i)
Yield 61%; light yellow oil; 1H NMR (500 MHz, CDCl3): d 7.78(s,
1H), 7.57(d, J = 8.7 Hz, 2H), 6.98(d, J = 8.7 Hz, 2H), 4.44(s, 2H), 3.86
(s, 3H), 3.85(s, 3H); 13C NMR (125 MHz, CDCl3): d 166.9, 160.8,
143.0, 132.0, 126.7, 126.1, 114.4, 55.4, 52.4, 27.6.
4.2.10. (Z)-Methyl 2-(bromomethyl)-3-(4-ethoxyphenyl)acrylate (3j)
Yield 51%; yellow oil; 1H NMR (500 MHz, CDCl3): d 7.77(s, 1H),
7.56(d, J = 8.8 Hz, 2H), 6.96(d, J = 8.8 Hz, 2H), 4.45(s, 2H), 4.08(q, J
= 7.0 Hz, 2H), 3.86(s, 3H), 1.44(t, J = 7.0 Hz, 3H); 13C NMR (125
MHz, CDCl3): d 167.0, 160.3, 143.0, 132.0, 126.5, 126.0, 114.9,
63.6, 52.3, 27.6, 14.7.
4.2.1. (Z)-Methyl 2-(bromomethyl)-3-phenylacrylate (3a)
Yield 75%; light yellow oil; 1H NMR (500 MHz, CDCl3): d 7.84(s,
1H), 7.58(d, J = 7.6 Hz, 2H), 7.41–7.49(overlapped, 3H), 4.40(s, 2H),
3.89(s, 3H); 13C NMR (125 MHz, CDCl3): d 166.6, 143.0, 134.2,
129.6, 128.9, 128.6, 52.5, 26.8.
4.2.11. (Z)-Methyl 2-(bromomethyl)-3-(3-chlorophenyl)acrylate (3k)
Yield 77%; yellow oil; 1H NMR (500 MHz, CDCl3): d 7.73(s, 1H),
7.69(s, 1H), 7.52(t, J = 8.4 Hz, 2H), 7.33(t, J = 8.4 Hz, 1H), 4.34(s,
2H), 3.88(s, 3H); 13C NMR (125 MHz, CDCl3): d 166.2, 141.1,
136.2, 132.5, 132.3, 130.4, 130.1, 127.8, 122.9, 52.6, 26.0.
4.2.2. (Z)-Methyl 2-(bromomethyl)-3-(4-fluorophenyl)acrylate (3b)
Yield 79%; light yellow oil; 1H NMR (500 MHz, CDCl3): d 7.78(s,
1H), 7.57–7.60(m, 2H), 7.14–7.17(m, 2H), 4.37(s, 2H), 3.88(s, 3H);
13C NMR (125 MHz, CDCl3): d 166.5, 163.0(d, J = 251.9 Hz), 141.8,
131.9(d, J = 8.1 Hz), 130.3(d, J = 3.5 Hz), 128.4, 116.1(d, J = 21.7
Hz), 52.5, 26.5.
4.2.12. (Z)-Methyl 2-(bromomethyl)-3-(m-tolyl)acrylate (3l)
Yield 63%; yellow oil; 1H NMR (500 MHz, CDCl3): d 7.81(s, 1H),
7.34–7.41(m, 3H), 7.23(d, J = 7.5 Hz, 1H), 4.41(s, 2H), 3.89(s, 3H),
2.41(s, 3H); 13C NMR (125 MHz, CDCl3): d 166.7, 143.2, 138.6,
134.2, 130.3, 128.8, 128.4, 126.6, 52.4, 26.9, 21.4.
4.2.13. (Z)-Methyl 2-(bromomethyl)-3-(2-chlorophenyl)acrylate (3m)
Yield 71%; yellow oil; 1H NMR (500 MHz, CDCl3): d 7.88(s, 1H),
7.71(d, J = 7.4 Hz, 1H), 7.67(d, J = 8.3 Hz, 1H), 7.46(t, J = 7.5 Hz, 2H),
7.30(t, J = 8.2 Hz, 1H), 4.28(s, 2H), 3.93(s, 3H); 13C NMR (125 MHz,
CDCl3): d 166.1, 141.7, 134.7, 133.0, 130.7, 130.3, 129.6, 127.6,
124.4, 52.6, 26.2.
4.2.3. (Z)-Methyl 2-(bromomethyl)-3-(4-chlorophenyl)acrylate (3c)
Yield 67%; light yellow oil; 1H NMR (500 MHz, CDCl3): d 7.77(s,
1H), 7.52(d, J = 7.8 Hz, 2H), 7.44(d, J = 7.8 Hz, 2H), 4.36(s, 2H), 3.89
(s, 3H); 13C NMR (125 MHz, CDCl3): d 166.4, 141.5, 135.7, 132.6,
130.8, 129.2, 129.1, 52.6, 26.3.
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W. Delong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.2.14. (Z)-Methyl 2-(bromomethyl)-3-(o-tolyl)acrylate (3n)
Yield 64%; yellow oil; 1H NMR (500 MHz, CDCl3): d 7.95(s, 1H),
7.58–7.60(m, 1H), 7.27–7.34(m, 3H), 4.32(s, 2H), 3.93(s, 3H), 2.34
(s, 3H); 13C NMR (125 MHz, CDCl3): d 166.5, 142.1, 137.3, 133.5,
130.3, 129.5, 129.4, 128.0, 126.1, 52.4, 26.6, 19.9.
168.7, 141.8, 139.0, 132.3, 130.1, 129.7, 129.2, 51.9, 51.5, 45.9,
23.0, 21.3, 11.8.
4.3.6. (E)-Methyl 3-(4-ethylphenyl)-2-((propylamino)methyl)acrylate
(4f)
Yield 49%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.78(s,
1H), 7.42(d, J = 7.9 Hz, 2H), 7.21(d, J = 7.9 Hz, 2H), 3.80(s, 3H),
3.59(s, 2H), 2.65(q, J = 7.9 Hz, 2H), 2.59(t, J = 7.2 Hz, 2H), 1.67(s,
1H), 1.47–1.54(m, 2H), 1.24(t, J = 7.2 Hz, 3H), 0.92(t, J = 7.2 Hz,
3H); 13C NMR (125 MHz, CDCl3): d 168.7, 145.3, 141.8, 132.6,
130.1, 129.8, 128.0, 51.9, 51.5, 45.9, 28.7, 23.0, 15.3, 11.8.
4.2.15. (Z)-Methyl 2-(bromomethyl)-3-(4-nitrophenyl)acrylate (3o)
Yield 57%; yellow oil; 1H NMR (500 MHz, CDCl3): d 8.30(d, J =
8.8 Hz, 2H), 7.82(s, 1H), 7.71(d, J = 8.4 Hz, 2H), 4.30(s, 2H), 3.90(s,
3H); 13C NMR (125 MHz, CDCl3): d 165.8, 147.9, 140.5, 139.8,
131.9, 130.1, 124.0, 52.8, 25.3.
4.2.16. (Z)-Methyl 2-(bromomethyl)-3-(naphthalen-2-yl)acrylate (3p)
Yield 79%; yellow oil; 1H NMR (500 MHz, CDCl3): d 8.11(s, 1H),
7.99(s, 1H), 7.85–7.91(m, 3H), 7.52–7.66(m, 3H), 4.49(s, 2H), 3.92
(s, 3H); 13C NMR (125 MHz, CDCl3): d 166.7, 143.1, 133.5, 133.1,
130.1, 128.7, 128.6, 127.7, 127.5, 126.8, 126.4, 52.5, 27.1.
4.3.7. (E)-Methyl 3-(4-isopropylphenyl)-2-((propylamino)methyl)
acrylate (4g)
Yield 47%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.82(s,
1H), 7.48(d, J = 7.9 Hz, 2H), 7.29(d, J = 7.9 Hz, 2H), 3.85(s, 3H),
3.64(s, 2H), 2.92–3.00(m, 1H), 2.64(t, J = 7.2 Hz, 2H), 1.79(s, 1H),
1.51–1.59(m, 2H), 1.24(d, J = 7.2 Hz, 6H), 0.96(t, J = 7.2 Hz, 3H);
13C NMR (125 MHz, CDCl3): d 168.7, 149.5, 141.8, 132.7, 130.0,
129.8, 126.6, 51.9, 51.5, 45.9, 34.0, 23.8, 23.0, 11.8.
4.3. Typical procedure for synthesis of compounds 4a–p and 6a–m36
A solution of 3 (1.0 mmol) in dry CH3CN (1.0 mL) was dropped
into a mixture of amine (aliphatic amine 3.0 mmol, aromatic amine
1.0 mmol) and powdered K2CO3 (3.0 mmol) in 6.0 mL dry CH3CN.
After stirring at r.t. for 1 h for aliphatic amine or 3 h for aromatic
amine, the mixture was filtered and the filtrate was evaporated.
The target products were obtained by column chromatography
with petroleum ether/EtOAC as eluent (15/2 for aliphatic amine
product, 15/1 for aromatic amine product).
4.3.8. (E)-Methyl 3-(4-(tert-butyl)phenyl)-2-((propylamino)methyl)
acrylate (4h)
Yield 51%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.78(s,
1H), 7.45(d, J = 8.5 Hz, 2H), 7.41(d, J = 8.5 Hz, 2H), 3.82(s, 3H),
3.61(s, 2H), 2.61(t, J = 6.4 Hz, 2H), 1.66(s, 1H), 1.48–1.66(m, 2H),
1.33(s, 9H), 0.93(t, J = 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3): d
168.7, 152.2, 141.7, 132.3, 130.1, 129.6, 125.4, 51.9, 51.5, 45.9,
34.7, 31.2, 23.1, 11.8.
4.3.1. (E)-Methyl 3-phenyl-2-((propylamino)methyl)acrylate (4a)
Yield 69%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.80(s,
1H), 7.48(d, J = 7.8 Hz, 2H), 7.33–7.40(overlapped, 3H), 3.82(s,
3H), 3.58(s, 2H), 2.57(t, J = 7.5 Hz, 2H), 1.74(s, 1H), 1.47–1.52(m,
2H), 0.91(t, J = 7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 168.6,
141.7, 135.2, 131.0, 129.5, 128.8, 128.4, 52.0, 51.4, 45.8, 23.0, 11.7.
4.3.9. (E)-Methyl 3-(4-methoxyphenyl)-2-((propylamino)methyl)
acrylate (4i)
Yield 41%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.74(s,
1H), 7.49(d, J = 7.8 Hz, 2H), 6.90(d, J = 8.5 Hz, 2H), 3.81(s, 3H),
3.79(s, 3H), 3.58(s, 2H), 2.60(t, J = 6.2 Hz, 2H), 1.72(s, 1H), 1.48–
1.55(m, 2H), 0.92(t, J = 7.8 Hz, 3H); 13C NMR (125 MHz, CDCl3): d
168.8, 160.2, 141.6, 131.5, 128.6, 127.7, 113.9, 55.2, 51.5, 51.6,
46.0, 23.0, 11.8.
4.3.2. (E)-Methyl 3-(4-fluorophenyl)-2-((propylamino)methyl)
acrylate (4b)
Yield 47%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.74(s,
1H), 7.50–7.53(m, 2H), 7.04–7.08(m, 2H), 3.80(s, 3H), 3.52(s, 2H),
2.58(t, J = 7.1 Hz, 2H), 1.57(s, 1H), 1.47–1.51(m, 2H), 0.91(t, J =
7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 168.5, 162.9(d, J =
253.2 Hz), 140.6, 131.7(d, J = 8.6 Hz), 131.3(d, J = 3.6 Hz), 130.7,
115.5(d, J = 21.5 Hz), 52.0, 51.6, 45.9, 23.0, 11.7.
4.3.10. (E)-Methyl 3-(4-ethoxyphenyl)-2-((propylamino)methyl)
acrylate (4j)
Yield 42%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.74(s,
1H), 7.47(d, J = 8.1 Hz, 2H), 6.89(d, J = 8.1 Hz, 2H), 4.03(t, J = 6.8
Hz, 2H), 3.79(s, 3H), 3.58(s, 2H), 2.60(t, J = 6.2 Hz, 2H), 1.76(s,
1H), 1.49–1.53(m, 2H), 1.40(t, J = 6.8 Hz, 3H), 0.92(t, J = 7.5 Hz,
3H); 13C NMR (125 MHz, CDCl3): d 168.8, 159.6, 141.7, 131.5,
128.6, 127.5, 114.4, 63.4, 51.9, 51.6, 46.0, 23.0, 14.7, 11.8.
4.3.3. (E)-Methyl 3-(4-chlorophenyl)-2-((propylamino)methyl)
acrylate (4c)
Yield 51%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.70(s,
1H), 7.74(d, J = 8.5 Hz, 2H), 7.33(d, J = 8.5 Hz, 2H), 3.79(s, 3H),
3.49(s, 2H), 2.55(t, J = 7.1 Hz, 2H), 1.58(s, 1H), 1.44–1.51(m, 2H),
0.90(t, J = 7.6 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 168.3, 140.4,
134.8, 133.6, 131.4, 131.0, 128.7, 52.0, 51.6, 45.9, 23.0, 11.7.
4.3.11. (E)-Methyl 3-(3-chlorophenyl)-2-((propylamino)methyl)
acrylate (4k)
Yield 55%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.78(s,
1H), 7.73(s, 1H), 7.49(d, J = 6.9 Hz, 1H), 7.45(d, J = 6.9 Hz, 1H),
7.28(d, J = 8.1 Hz, 1H), 3.85(s, 3H), 3.54(s, 2H), 2.63(t, J = 6.2 Hz,
2H), 1.63(s, 1H), 1.49–1.53(m, 2H), 0.97(t, J = 6.8 Hz, 3H); 13C
NMR (125 MHz, CDCl3): d 168.2, 140.0, 137.2, 132.5, 132.2, 131.7,
129.9, 128.1, 122.6, 52.1, 51.5, 46.8, 23.0, 11.8.
4.3.4. (E)-Methyl 3-(4-bromophenyl)-2-((propylamino)methyl)
acrylate (4d)
Yield 50%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.70(s,
1H), 7.51(d, J = 8.5 Hz, 2H), 7.40(d, J = 8.2 Hz, 2H), 3.82(s, 3H),
3.51(s, 2H), 2.57(t, J = 7.2 Hz, 2H), 1.59(s, 1H), 1.46–1.53(m, 2H),
0.92(t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 168.3, 140.5,
134.1, 131.7, 131.5, 131.2, 123.2, 52.1, 51.6, 45.9, 23.0, 11.8.
4.3.12. (E)-Methyl 2-((propylamino)methyl)-3-(m-tolyl)acrylate (4l)
Yield 59%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.81(s,
1H), 7.19–7.35(m, 4H), 3.85(s, 3H), 3.61(s, 2H), 2.61(t, J = 6.2 Hz,
2H), 2.40(s, 3H), 1.69(s, 1H), 1.52–1.55(m, 2H), 0.96(t, J = 7.5 Hz,
3H); 13C NMR (125 MHz, CDCl3): d 168.6, 141.9, 138.0, 135.2,
130.8, 130.2, 129.6, 128.3, 126.6, 51.9, 51.4, 45.9, 23.0, 21.4, 11.8.
4.3.5. (E)-Methyl 2-((propylamino)methyl)-3-(p-tolyl)acrylate (4e)
Yield 49%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.77(s,
1H), 7.39(d, J = 7.2 Hz, 2H), 7.19(d, J = 7.2 Hz, 2H), 3.80(s, 3H),
3.57(s, 2H), 2.58(t, J = 7.2 Hz, 2H), 2.35(s, 3H), 1.71(s, 1H), 1.46–
1.53(m, 2H), 0.91(t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3): d
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4.3.13. (E)-Methyl 3-(2-chlorophenyl)-2-((propylamino)methyl)
acrylate (4m)
4.3.21. (E)-Methyl 2-((sec-butylamino)methyl)-3-(4-chlorophenyl)
acrylate (6e)
Yield 51%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.81(s,
1H), 7.54–7.61(m, 2H), 7.18–7.34(m, 2H), 3.84(s, 3H), 3.47(s, 2H),
2.50(t, J = 7.0 Hz, 2H), 1.69(s, 1H), 1.39–1.46(m, 2H), 0.88(t, J =
7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 168.0, 140.6, 135.7,
132.7, 132.2, 130.9, 130.0, 127.2, 124.1, 52.1, 51.1, 45.8, 22.9, 11.7.
Yield 59%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.73(s,
1H), 7.51(d, J = 8.4 Hz, 2H), 7.36(d, J = 8.5 Hz, 2H), 3.82(s, 3H),
3.47–3.58(m, 2H), 2.54–2.61(m, 1H), 1.55(s, 1H), 1.42–1.50(m,
1H), 1.30–1.39(m, 1H), 1.03(d, J = 6.3 Hz, 3H), 0.90(t, J = 6.3 Hz,
3H); 13C NMR (125 MHz, CDCl3): d 168.4, 140.4, 134.9, 133.6,
131.5, 131.0, 128.6, 54.8, 52.0, 43.7, 29.4, 19.8, 10.2.
4.3.14. (E)-Methyl 2-((propylamino)methyl)-3-(o-tolyl)acrylate (4n)
Yield 50%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.86(s,
1H), 7.19–7.37(m, 4H), 3.83(s, 3H), 3.49(s, 2H), 2.48(t, J = 8.3 Hz,
2H), 2.3(s, 3H), 1.66(s, 1H), 1.38–1.45(m, 2H), 0.87(t, J = 7.4 Hz,
3H); 13C NMR (125 MHz, CDCl3): d 168.3, 140.7, 136.8, 134.5,
131.5, 130.0, 129.0, 128.6, 125.7, 51.9, 51.1, 45.7, 22.9, 19.9, 11.7.
4.3.22. (E)-Methyl 3-(4-chlorophenyl)-2-((hexylamino)methyl)
acrylate (6f)
Yield 41%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.73(s,
1H), 7.46(d, J = 8.4 Hz, 2H), 7.36(d, J = 8.4 Hz, 2H), 3.82(s, 3H),
3.52(s, 2H), 2.60(t, J = 7.6 Hz, 2H), 1.57(s, 1H), 1.44–1.50(m, 2H),
1.26–1.35(m, 6H), 0.89(d, J = 6.7 Hz, 3H); 13C NMR (125 MHz,
CDCl3): d 168.4, 140.4, 134.9, 133.7, 131.5, 131.0, 128.7, 52.1,
49.7, 45.9, 31.7, 29.9, 27.0, 22.6, 14.0.
4.3.15. (E)-Methyl 3-(4-nitrophenyl)-2-((propylamino)methyl)
acrylate (4o)
Yield 71%; colorless solid; m.p. 71–74 °C; 1H NMR (500 MHz,
CDCl3): d 8.25(d, J = 9.0 Hz, 2H), 7.79(s, 1H), 7.72(d, J = 8.5 Hz,
2H), 3.85(s, 3H), 3.50(s, 2H), 2.59(t, J = 6.8 Hz, 2H), 1.47–1.54(over-
lapped, 3H), 0.94(t, J = 7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3): d
167.9, 147.7, 141.7, 139.0, 134.3, 130.5, 123.7, 52.3, 51.7, 46.0,
23.0, 11.8.
4.3.23. (E)-Methyl 3-(4-chlorophenyl)-2-((cyclohexylamino)methyl)
acrylate (6g)
Yield 55%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.71(s,
1H), 7.49(d, J = 8.5 Hz, 2H), 7.35(d, J = 8.5 Hz, 2H), 3.81(s, 3H),
3.54(s, 2H), 2.42–2.47(m, 1H), 1.57–1.83(m, 5H), 1.50(s, 1H),
1.07–1.28(m, 5H); 13C NMR (125 MHz, CDCl3): d 168.3, 140.3,
134.8, 133.7, 131.5, 131.0, 128.6, 56.9, 52.0, 43.4, 33.4, 26.1, 24.9.
4.3.16. (E)-Methyl 3-(naphthalen-2-yl)-2-((propylamino)methyl)
acrylate (4p)
4.3.24. (E)-Methyl 3-(4-chlorophenyl)-2-((octylamino)methyl)
acrylate (6h)
Yield 41%; colorless oil; 1H NMR (500 MHz, CDCl3): d 8.05(s,
1H), 7.79(s, 1H), 7.83–7.87(m, 3H), 7.49–7.62(m, 3H), 3.87(s, 3H),
3.67(s, 2H), 2.65(t, J = 6.9 Hz, 2H), 1.65(s, 1H), 1.52–1.59(m, 2H),
0.97(t, J = 7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 168.7, 141.8,
133.3, 133.2, 132.8, 131.2, 129.6, 128.5, 128.1, 127.7, 127.7,
126.8, 126.4, 52.0, 51.7, 46.1, 23.1, 11.9.
Yield 57%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.71(s,
1H), 7.45(d, J = 8.6 Hz, 2H), 7.33(d, J = 8.1 Hz, 2H), 3.81(s, 3H),
3.50(s, 2H), 2.58(d, J = 6.7 Hz, 2H), 1.52(s, 1H), 1.43–1.52(m, 2H),
1.26–1.31(s, 10H), 0.87(t, J = 7.1 Hz, 3H); 13C NMR (125 MHz,
CDCl3): d 168.3, 140.3, 134.8, 133.6, 131.5, 131.0, 128.7, 52.0,
49.7, 45.9, 31.8, 29.9, 29.5, 29.3, 27.3, 22.6, 14.0.
4.3.17. (E)-Methyl 3-(4-chlorophenyl)-2-((isopropylamino)methyl)
acrylate (6a)
4.3.25. (E)-Methyl 3-(4-chlorophenyl)-2-((phenylamino)methyl)
acrylate (6i)
Yield 61%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.70(s,
1H), 7.48(d, J = 8.4 Hz, 2H), 7.34(d, J = 8.4 Hz, 2H), 3.80(s, 3H),
3.51(s, 2H), 2.81(q, J = 6.3 Hz, 1H), 1.59(s, 1H), 1.04(d, J = 6.4 Hz,
6H); 13C NMR (125 MHz, CDCl3): d 168.3, 140.3, 134.9, 133.6,
131.4, 131.0, 128.7, 52.0, 48.8, 43.8, 22.8.
Yield 38%; yellow solid; m.p. 112–114 °C; 1H NMR (500 MHz,
CDCl3): d 7.83(s, 1H), 7.37–7.41(m, 4H), 7.15–7.18(m, 2H), 6.75(d,
J = 7.3 Hz, 2H), 6.56(d, J = 7.6 Hz, 2H), 4.10(s, 2H), 4.05(s, 1H),
3.84(s, 3H); 13C NMR (125 MHz, CDCl3): d 167.9, 147.6, 141.6,
135.4, 133.2, 130.9, 129.7, 129.2, 129.0, 118.1, 113.5, 52.3, 41.0.
4.3.18. (E)-Methyl 2-((butylamino)methyl)-3-(4-chlorophenyl)
acrylate (6b)
4.3.26. (E)-Methyl 3-(4-chlorophenyl)-2-(((4-fluorophenyl)amino)
methyl)acrylate (6j)
Yield 69%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.73(s,
1H), 7.47(d, J = 8.4 Hz, 2H), 7.37(d, J = 8.4 Hz, 2H), 3.82(s, 3H),
3.52(s, 2H), 2.61(t, J = 7.7 Hz, 1H), 1.58(s, 1H), 1.44–1.50(m, 2H),
1.32–1.40(m, 2H), 0.92(t, J = 7.2 Hz, 3H); 13C NMR (125 MHz,
CDCl3): d 168.4, 140.4, 134.9, 133.6, 131.4, 131.0, 128.7, 52.1,
49.3, 45.9, 32.0, 20.4, 13.9.
Yield 41%; yellow solid; m.p. 95–99 °C; 1H NMR (500 MHz,
CDCl3): d 7.82(s, 1H), 7.36–7.39(m, 4H), 6.84–6.88(m, 2H), 6.45–
6.48(m, 2H), 4.05(s, 2H), 3.97(s, 1H), 3.84(s, 3H); 13C NMR (125
MHz, CDCl3): d 167.9, 156.2(d, J = 236.1 Hz), 143.9(d, J = 1.7 Hz),
141.6, 135.4, 133.1, 130.8, 129.7, 129.0, 115.6(d, J = 22.5 Hz),
114.4(d, J = 7.4 Hz), 52.3, 41.7.
4.3.19. (E)-Methyl 3-(4-chlorophenyl)-2-((isobutylamino)methyl)
acrylate (6c)
4.3.27. (E)-Methyl 3-(4-chlorophenyl)-2-(((4-chlorophenyl)amino)
methyl)acrylate (6k)
Yield 65%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.72(s,
1H), 7.49(d, J = 8.5 Hz, 2H), 7.35(d, J = 8.5 Hz, 2H), 3.82(s, 3H),
3.49(s, 2H), 2.43(d, J = 7.2 Hz, 2H), 1.68–1.76(m, 2H), 1.55(s, 1H),
0.92(d, J = 6.6 Hz, 6H); 13C NMR (125 MHz, CDCl3): d 168.4, 140.4,
134.9, 133.7, 131.5, 131.0, 128.7, 57.8, 52.0, 46.1, 28.2, 20.6.
Yield 37%; yellow solid; m.p. 117–119 °C; 1H NMR (500 MHz,
CDCl3): d 7.82(s, 1H), 7.35–7.40(m, 4H), 7.08–7.10(m, 2H), 6.42–
6.44(m, 2H), 4.07(overlapped, 3H), 3.84(s, 3H); 13C NMR (125
MHz, CDCl3): d 167.8, 146.1, 141.6, 135.5, 133.1, 130.8, 129.5,
129.0, 129.0, 122.7, 114.5, 52.3, 41.0.
4.3.20. (E)-Methyl 2-((tert-butylamino)methyl)-3-(4-chlorophenyl)
acrylate (6d)
4.3.28. (E)-Methyl 2-(((4-bromophenyl)amino)methyl)-3-(4-
chlorophenyl)acrylate (6l)
Yield 51%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.71(s,
1H), 7.56(d, J = 9.1 Hz, 2H), 7.34(d, J = 8.3 Hz, 2H), 3.81(s, 3H),
1.35(s, 1H), 1.14(s, 9H); 13C NMR (125 MHz, CDCl3): d 168.4,
140.4, 134.9, 133.7, 131.7, 131.1, 128.5, 52.0, 50.9, 39.6, 28.9.
Yield 37%; yellow solid; m.p. 129–131 °C; 1H NMR (500 MHz,
CDCl3): d 7.83(s, 1H), 7.34–7.40(m, 4H), 7.21–7.23(m, 2H), 6.37–
6.39(m, 2H), 4.10(s, 1H), 4.07(s, 2H), 3.84(s, 3H); 13C NMR (125
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W. Delong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
MHz, CDCl3): d 167.8, 146.5, 141.7, 135.5, 133.1, 131.9, 130.8,
129.4, 129.0, 115.0, 109.7, 52.3, 40.9.
2H), 3.33(t, J = 7.0 Hz, 2H), 2.65(q, J = 7.6 Hz, 2H), 1.59–1.66(m,
2H), 1.23(t, J = 7.5 Hz, 3H), 0.96(t, J = 7.5 Hz, 3H); 13C NMR (125
MHz, CDCl3): d 164.9, 145.6, 135.6, 131.9, 128.6, 128.4, 123.3,
49.0, 43.7, 28.7, 21.3, 15.3, 11.5; ESI-MS: m/z 229.0 ([M+H]+).
4.3.29. (E)-Methyl 3-(4-chlorophenyl)-2-((p-tolylamino)methyl)
acrylate (6m)
Yield 41%; yellow oil; 1H NMR (500 MHz, CDCl3): d 7.83(s, 1H),
7.37–7.43(m, 4H), 6.99(d, J = 8.1 Hz, 2H), 6.50(d, J = 8.5 Hz, 2H),
4.08(s, 2H), 3.85(s, 3H), 2.27(s, 3H); 13C NMR (125 MHz, CDCl3): d
168.0, 145.3, 141.5, 135.3, 133.2, 130.9, 129.9, 129.7, 129.0,
127.4, 113.7, 52.3, 41.4, 20.4.
4.4.7. (E)-3-(4-Isopropylbenzylidene)-1-propylazetidin-2-one (5g)
Yield 56%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.24–7.30
(overlapped, 4H), 6.93(s, 1H), 4.12(d, J = 1.1 Hz, 2H), 3.37(t, J = 7.1
Hz, 2H), 2.90–2.98(m, 1H), 1.63–1.70(m, 2H), 1.27(d, J = 7.0 Hz,
6H), 0.99(t, J = 7.6 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 164.9,
150.2, 135.6, 132.0, 128.6, 127.0, 123.3, 49.0, 43.7, 32.0, 23.8,
21.3, 11.5; ESI-MS: m/z 243.0 ([M+H]+).
4.4. Typical procedure for synthesis of compounds 5a–p and 7a–c, e–
m
4.4.8. (E)-3-(4-(Tert-butyl)benzylidene)-1-propylazetidin-2-one (5h)
Yield 61%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.39(d, J =
8.5 Hz, 2H), 7.26(d, J = 8.5 Hz, 2H), 6.91(s, 1H), 4.09(d, J = 1.3 Hz,
2H), 3.35(t, J = 7.2 Hz, 2H), 1.60–1.68(m, 2H), 1.32(s, 9H), 0.97(t, J
= 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 164.9, 152.4, 135.8,
131.7, 128.3, 125.9, 123.2, 49.0, 43.7, 34.7, 31.1, 21.3, 11.5; ESI-
MS: m/z 257.0 ([M+H]+).
The intermediate 4 or 6 (1.0 eq) was dissolved in dry THF (1.0
mmol/20 mL) under nitrogen atmosphere and cooled to ꢀ20 °C.
To the above stirring solution was added lithium bis(trimethylsi-
lyl)amide(LHMDS) (1.0 M in THF) (1.5 eq) and the resulting mix-
ture was stirred at ꢀ20 °C for 1 h. Then, distilled water was
added to quench the reaction and the solution was evaporated to
remove THF. The residual was dissolved in EtOAc, washed with dis-
tilled water for 3 times, dried over Na2SO4, and evaporated. The
crude product was purified by column chromatography (petro-
leum ether/EtOAc, 10/1).
4.4.9. (E)-3-(4-Methoxybenzylidene)-1-propylazetidin-2-one (5i)
Yield 47%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.25(d, J =
7.8 Hz, 2H), 6.86–6.89(overlapped, 3H), 4.06(d, J = 1.3 Hz, 2H), 3.81
(s, 3H), 3.33(t, J = 6.9 Hz, 2H), 1.58–1.66(m, 2H), 0.96(t, J = 7.5 Hz,
3H); 13C NMR (125 MHz, CDCl3): d 165.1, 160.3, 134.2, 130.0,
127.1, 122.9, 114.4, 55.3, 48.9, 43.7, 21.3, 11.5; ESI-MS: m/z 231.0
([M+H]+).
4.4.1. (E)-3-Benzylidene-1-propylazetidin-2-one (5a)36
Yield 74%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.30–7.37
(overlapped, 5H), 6.91(s, 1H), 4.09(d, J = 1.2 Hz, 2H), 3.34(t, J = 7.1
Hz, 2H), 1.59–1.67(m, 2H), 0.96(t, J = 7.4 Hz, 3H); 13C NMR (125
MHz, CDCl3): d 164.7, 136.6, 134.4, 129.0, 128.9, 128.5, 123.3,
49.0, 43.7, 21.2, 11.5; ESI-MS: m/z 202.0 ([M+H]+).
4.4.10. (E)-3-(4-Ethoxybenzylidene)-1-propylazetidin-2-one (5j)
Yield 44%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.24(d, J =
9.2 Hz, 2H), 6.86–6.89(overlapped, 3H), 4.02–4.07(overlapped, 4H),
3.34(t, J = 6.2 Hz, 2H), 1.59–1.66(m, 2H), 1.42(t, J = 7.0 Hz, 3H), 0.96
(t, J = 7.7 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 165.1, 159.7, 134.0,
130.0, 127.0, 123.0, 114.9, 63.5, 48.9, 43.7, 21.3, 14.7, 11.5; ESI-MS:
m/z 245.0 ([M+H]+).
4.4.2. (E)-3-(4-Fluorobenzylidene)-1-propylazetidin-2-one (5b)
Yield 65%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.26–7.29
(m, 2H), 7.02–7.05(m, 2H), 6.86(s, 1H), 4.06(d, J = 1.0 Hz, 2H), 3.33
(t, J = 7.1 Hz, 2H), 1.59–1.66(m, 2H), 0.95(t, J = 7.4 Hz, 3H); 13C NMR
(125 MHz, CDCl3): d 164.5, 163.0(d, J = 251.2 Hz), 136.3(d, J = 2.0
Hz), 130.6(d, J = 3.5 Hz), 130.3(d, J = 8.3 Hz), 122.1, 116.0(d, J =
21.7 Hz),48.8, 43.7, 21.2, 11.6; ESI-MS: m/z 220.0 ([M+H]+).
4.4.11. (E)-3-(3-Chlorobenzylidene)-1-propylazetidin-2-one (5k)
Yield 59%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.42–7.45
(overlapped, 2H), 7.23(d, J = 4.9 Hz, 2H), 6.83(s, 1H), 4.09(d, J = 1.3
Hz, 2H), 3.35(t, J = 7.0 Hz, 2H), 1.60–1.67(m, 2H), 0.96(t, J = 7.4 Hz,
3H); 13C NMR (125 MHz, CDCl3): d 164.1, 138.2, 136.5, 131.9,
131.0, 130.4, 127.1, 123.0, 121.8, 48.9, 43.8, 21.2, 11.5; ESI-MS:
m/z 236.0 ([M+H]+).
4.4.3. (E)-3-(4-Chlorobenzylidene)-1-propylazetidin-2-one (5c)
Yield 47%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.37(d, J =
8.5 Hz, 2H), 7.29(d, J = 8.5 Hz, 2H), 6.92(s, 1H), 4.12(d, J = 1.3 Hz,
2H), 3.39(t, J = 7.1 Hz, 2H), 1.66–1.72(m, 2H), 1.01(t, J = 7.4 Hz,
3H); 13C NMR (125 MHz, CDCl3): d 164.4, 137.3, 135.0, 132.9,
129.6, 129.2, 122.1, 48.9, 43.8, 21.2, 11.5; ESI-MS: m/z 236.0 ([M
+H]+).
4.4.12. (E)-3-(3-Methylbenzylidene)-1-propylazetidin-2-one (5l)
Yield 61%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.23–7.26
(m, 1H), 7.11–7.14(m, 3H), 6.88(s, 1H), 4.09(d, J = 1.3 Hz, 2H), 3.34
(t, J = 6.8 Hz, 2H), 2.35(s, 3H), 1.60–1.67(m, 2H), 0.97(t, J = 7.7 Hz,
3H); 13C NMR (125 MHz, CDCl3): d 164.8, 138.6, 136.4, 134.4,
129.9, 129.3, 128.8, 125.5, 123.5, 49.0, 43.7, 21.4, 21.3, 11.5; ESI-
MS: m/z 215.0 ([M+H]+).
4.4.4. (E)-3-(4-Bromobenzylidene)-1-propylazetidin-2-one (5d)
Yield 43%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.70(s,
1H), 7.47(d, J = 8.5 Hz, 2H), 7.15(d, J = 8.2 Hz, 2H), 6.83(s, 1H),
4.06(d, J = 1.3 Hz, 2H), 3.33(t, J = 7.3 Hz, 2H), 1.58–1.66(m, 2H),
0.95(t, J = 7.7 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 164.3, 137.5,
133.3, 132.1, 129.8, 123.2, 122.1, 48.9, 43.8, 21.2, 11.5; ESI-MS:
m/z 280.0 ([M+H]+).
4.4.13. (E)-3-(2-Chlorobenzylidene)-1-propylazetidin-2-one (5m)
Yield 63%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.58–7.60
(m, 1H), 7.14–7.32(overlapped, 4H), 4.05(d, J = 1.2 Hz, 2H), 3.34(t, J
= 7.7 Hz, 2H), 1.59–1.66(m, 2H), 0.96(t, J = 7.4 Hz, 3H); 13C NMR
(125 MHz, CDCl3): d 164.0, 139.1, 134.1, 133.6, 130.1, 128.1,
127.5, 125.4, 122.2, 48.6, 43.8, 21.2, 11.5; ESI-MS: m/z 236.0 ([M
+H]+).
4.4.5. (E)-3-(4-Methylbenzylidene)-1-propylazetidin-2-one (5e)
Yield 51%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.16–7.22
(overlapped, 4H), 6.90(s, 1H), 4.09(d, J = 1.3 Hz, 2H), 3.33(t, J = 7.0
Hz, 2H), 2.36(s, 3H), 1.62–1.66(m, 2H), 0.97(t, J = 7.3 Hz, 3H); 13C
NMR (125 MHz, CDCl3): d 164.9, 139.2, 135.6, 131.7, 129.6, 128.5,
123.3, 49.0, 43.7, 21.3, 21.3, 11.5; ESI-MS: m/z 215.0 ([M+H]+).
4.4.14. (E)-3-(2-Methylbenzylidene)-1-propylazetidin-2-one (5n)
Yield 57%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.17–7.28
(m, 5H), 4.08(d, J = 1.3 Hz, 2H), 3.37(t, J = 7.5 Hz, 2H), 2.4(s, 3H),
1.62–1.69(m, 2H), 0.99(t, J = 7.4 Hz, 3H); 13C NMR (125 MHz,
4.4.6. (E)-3-(4-Ethylbenzylidene)-1-propylazetidin-2-one (5f)
Yield 57%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.23(d, J =
8.0 Hz, 2H), 7.18(d, J = 8.0 Hz, 2H), 6.89(s, 1H), 4.08(d, J = 1.2 Hz,
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11
CDCl3): d 164.8, 137.7, 137.0, 132.9, 130.8, 129.0, 126.7, 126.1,
4.4.23. (E)-3-(4-Chlorobenzylidene)-1-octylazetidin-2-one (7h)
120.9, 48.9, 43.7, 21.2, 19.8, 11.5; ESI-MS: m/z 215.0 ([M+H]+).
Yield 61%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.33(d, J =
8.9 Hz, 2H), 7.23(d, J = 8.9 Hz, 2H), 6.87(s, 1H), 4.07(d, J = 1.3 Hz,
2H), 3.37(d, J = 7.5 Hz, 2H), 1.57–1.63(m, 2H), 1.27–1.34(s, 10H),
0.88(t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 164.2, 137.3,
134.9, 133.0, 128.6, 129.1, 122.0, 48.8, 42.1, 31.7, 29.1, 29.1, 27.8,
27.0, 22.6, 14.0; ESI-MS: m/z 306.1 ([M+H]+).
4.4.15. (E)-3-(4-Nitrobenzylidene)-1-propylazetidin-2-one (5o)36
Yield 56%; colorless solid; m.p. 73–76 °C; 1H NMR (500 MHz,
CDCl3): d 8.16–8.23(m, 4H), 6.40(s, 1H), 3.83(d, J = 1.3 Hz, 2H),
3.38(t, J = 7.4 Hz, 2H), 1.62–1.69(m, 2H), 0.94(t, J = 7.4 Hz, 3H);
13C NMR (125 MHz, CDCl3): d 161.9, 147.5, 140.9, 140.4, 130.4,
125.8, 123.8, 47.3, 44.0, 21.0, 11.5; ESI-MS: m/z 247.1 ([M+H]+).
4.4.24. (E)-3-(4-Chlorobenzylidene)-1-phenylazetidin-2-one (7i)
Yield 39%; colorless solid; m.p. 134–137 °C; 1H NMR (500 MHz,
CDCl3): d 7.12–7.45(overlapped, 10H), 4.05(d, J = 1.3 Hz, 2H); 13C
NMR (125 MHz, CDCl3): d 161.2, 138.5, 134.6, 134.2, 129.5, 129.3,
129.0, 128.8, 125.7, 123.9, 116.2, 48.5; ESI-MS: m/z 270.0 ([M+H]+).
4.4.16. (E)-3-(Naphthalen-2-ylmethylene)-1-propylazetidin-2-one
(5p)
Yield 51%; colorless solid; m.p. 69–71 °C; 1H NMR (500 MHz,
CDCl3): d 7.78–7.85(m, 4H), 7.38–7.51(m, 3H), 7.09(s, 1H), 4.19(d,
J = 1.3 Hz, 2H), 3.38(t, J = 6.9 Hz, 2H), 1.63–1.70(m, 2H), 0.99(t, J =
7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 164.7, 136.8, 133.4,
132.0, 129.3, 128.6, 128.2, 127.7, 126.9, 126.7, 124.7, 123.5, 49.1,
43.8, 21.3, 11.6; ESI-MS: m/z 252.0 ([M+H]+).
4.4.25. (E)-3-(4-Chlorobenzylidene)-1-(4-fluorophenyl)azetidin-2-one
(7j)
Yield 41%; colorless solid; m.p. 121–123 °C; 1H NMR (500 MHz,
CDCl3): d 7.82(s, 1H), 7.31–7.42(overlapped, 6H), 7.06–7.09(over-
lapped, 3H), 4.45(s, 2H); 13C NMR (125 MHz, CDCl3): d 160.6,
159.2(d, J = 244.5 Hz), 135.6, 135.0, 134.7(d, J = 2.3 Hz), 132.5,
129.8, 129.4, 124.5, 117.6(d, J = 7.9 Hz), 116.0(d, J = 22.7 Hz),
48.6; ESI-MS: m/z 288.0 ([M+H]+).
4.4.17. (E)-3-(4-Chlorobenzylidene)-1-isopropylazetidin-2-one (7a)
Yield 69%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.33(d, J =
8.7 Hz, 2H), 7.25(d, J = 8.4 Hz, 2H), 6.87(s, 1H), 4.08(q, J = 6.8 Hz,
1H), 4.05(d, J = 1.3 Hz, 2H), 1.04(d, J = 6.4 Hz, 6H); 13C NMR (125
MHz, CDCl3): d 163.4, 136.6, 134.9, 133.0, 129.6, 129.1, 122.0,
45.6, 43.7, 20.5; ESI-MS: m/z 236.0 ([M+H]+).
4.4.26. (E)-3-(4-Chlorobenzylidene)-1-(4-chlorophenyl)azetidin-2-
one (7k)
Yield 37%; colorless solid; m. p. 139–142 °C; 1H NMR (500 MHz,
CDCl3): d 7.31–7.40(m, 8H), 7.08(s, 1H), 4.46(d, J = 1.4 Hz, 2H); 13
C
4.4.18. (E)-1-Butyl-3-(4-chlorobenzylidene)azetidin-2-one (7b)
Yield 67%; colorless solid; m.p. 87–89 °C; 1H NMR (500 MHz,
CDCl3): d 7.33(d, J = 8.4 Hz, 2H), 7.24(d, J = 8.4 Hz, 2H), 6.87(s,
1H), 4.08(d, J = 1.3 Hz, 2H), 3.38(t, J = 7.7 Hz, 1H), 1.57–1.62(m,
2H), 1.35–1.43(m, 2H), 0.95(t, J = 7.2 Hz, 3H); 13C NMR (125 MHz,
CDCl3): d 164.3, 137.3, 134.9, 133.0, 129.6, 129.1, 122.0, 48.8,
41.8, 29.9, 20.2, 13.6; ESI-MS: m/z 250.0 ([M+H]+).
NMR (125 MHz, CDCl3): d 160.8, 136.9, 135.7, 134.8, 132.4, 129.9,
129.4, 129.4, 129.0, 124.8. 117.4. 48.6; ESI-MS: m/z 304.1 ([M+H]+).
4.4.27. (E)-3-(4-Bromobenzylidene)-1-(4-chlorophenyl)azetidin-2-
one (7l)
Yield 36%; colorless solid; m.p. 125–127 °C; 1H NMR (500 MHz,
CDCl3): d 7.30–7.49(m, 8H), 7.08(s, 1H), 4.45(d, J = 1.4 Hz, 2H); 13
C
NMR (125 MHz, CDCl3): d 160.8, 137.4, 135.7, 134.8, 132.4, 132.3,
129.9, 129.4, 124.9, 117.7, 116.5, 48.5; ESI-MS: m/z 348.0 ([M+H]+).
4.4.19. (E)-3-(4-Chlorobenzylidene)-1-isobutylazetidin-2-one (7c)
Yield 71%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.33(d, J =
8.5 Hz, 2H), 7.23(d, J = 8.5 Hz, 2H), 6.87(s, 1H), 4.10(d, J = 1.3 Hz,
2H), 3.19(d, J = 7.2 Hz, 2H), 1.90–1.98(m, 2H), 1.55(s, 1H), 0.97(d,
J = 6.6 Hz, 6H); 13C NMR (125 MHz, CDCl3): d 164.6, 137.4, 134.9,
132.9, 129.6, 129.2, 122.1, 49.9, 49.9, 27.8, 20.3; ESI-MS: m/z
250.0 ([M+H]+).
4.4.28. (E)-1-(4-Chlorophenyl)-3-(4-methylbenzylidene)azetidin-2-
one (7m)
Yield 41%; colorless solid; m.p. 143–145 °C; 1H NMR (500 MHz,
CDCl3): d 7.16–7.39(m, 8H), 7.03(s, 1H), 4.42(d, J = 1.3 Hz, 2H), 2.33
(s, 3H); 13C NMR (125 MHz, CDCl3): d 160.7, 136.0, 135.4, 135.3,
133.7, 132.7, 129.8, 129.8, 129.3, 123.9, 116.2, 48.4, 20.9; ESI-MS:
m/z 284.0 ([M+H]+).
4.4.20. (E)-1-(Sec-butyl)-3-(4-chlorobenzylidene)azetidin-2-one (7e)
Yield 57%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.31(d, J =
8.4 Hz, 2H), 7.23(d, J = 8.5 Hz, 2H), 6.86(s, 1H), 4.04(dd, J = 7.6, 1.3
Hz, 1H), 4.00(dd, J = 7.6, 1.3 Hz, 1H), 3.82–3.87(m, 1H), 1.52–1.62
(m, 2H), 1.22(d, J = 6.9 Hz, 3H), 0.94(t, J = 7.3 Hz, 3H); 13C NMR
(125 MHz, CDCl3): d 164.0, 136.6, 134.9, 133.0, 129.6, 129.1,
121.9, 49.4, 45.7, 27.8, 18.3, 10.9; ESI-MS: m/z 250.0 ([M+H]+).
4.5. Antifungal activity assay
The test compounds were dissolved in dimethyl sulfoxide
(DMSO) to prepare the stock solutions. Potato glucose agar (PDA)
culture medium were autoclaved and cooled before use. Appropri-
ate amounts of the test compound stocks were added rapidly to
thawed PDA culture medium (30 mL) under 50 °C to afford final
desired concentrations. After mixing homogeneously, the mixtures
poured in Petri dishes (9 cm in diameter). After the medium were
cooled and solidified, each plate was incubated with 4 mm myce-
lium disk at its center, inverted, and incubated at 28 °C for 48 h.
The PDA medium without stock solution added to it was used as
the control. Three replicate plates for each concentration of a test
compound for every fungus were carried out. After the fungus
had grown on control plates close to the margin of plate, mycelial
growth radius of the treated fungus was recorded as the mean of
two diameters at cross direction. The fungal growth inhibition
rates were calculated according to the following formula: Growth
inhibition rate (I) = [(C ꢀ T)/C] ꢂ 100%. Herein, C is the mycelial
growth radius of the control group, T is the mycelial growth radius
of the treatment group. After conversion of inhibition rates into
4.4.21. (E)-3-(4-Chlorobenzylidene)-1-hexylazetidin-2-one (7f)
Yield 59%; colorless oil; 1H NMR (500 MHz, CDCl3): d 7.33(d, J =
8.4 Hz, 2H), 7.23(d, J = 8.4 Hz, 2H), 6.87(s, 1H), 4.07(d, J = 1.3 Hz,
2H), 3.37(t, J = 7.6 Hz, 2H), 1.57–1.63(m, 2H), 1.31–1.37(m, 6H),
0.89(d, J = 6.7 Hz, 3H); 13C NMR (125 MHz, CDCl3): d 164.3, 137.3,
134.9, 133.0, 129.6, 129.1, 122.0, 48.8, 42.1, 31.4, 27.8, 26.7, 22.5,
13.9; ESI-MS: m/z 278.1 ([M+H]+).
4.4.22. (E)-3-(4-Chlorobenzylidene)-1-cyclohexylazetidin-2-one (7g)
Yield 55%; colorless oil; 1H NMR (500 MHz, CDCl3): d7.33(d, J =
8.6 Hz, 2H), 7.23(d, J = 8.6 Hz, 2H), 6.86(s, 1H), 4.06(d, J = 1.3 Hz,
2H), 3.66–3.72(m, 1H), 1.64–1.95(m, 5H), 1.15–1.45(m, 5H); 13C
NMR (125 MHz, CDCl3): d 163.4, 136.8, 134.9, 133.0, 129.6, 129.1,
121.9, 51.3, 46.3, 30.9, 25.3, 24.8; ESI-MS: m/z 276.1 ([M+H]+).
Please cite this article in press as: Delong W., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.003

12
W. Delong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
their probability values, the toxicity regression curve of probability
values versus corresponding logarithmic concentrations was plot-
ted. The EC50 (50% maximal effective concentration) of each test
compound was determined by the linear regression equation.
procedures previously reported by Tripti Singh et al.59 All the har-
vested specimen were fixed in 2.5% glutaraldehyde at 4 °C for 4 h,
then washed with 0.2 M PBS (phosphate buffered saline, pH 7.2; 3
ꢂ 15 min), and successively post-fixed in 1% OsO4 at 4 °C in the
same buffer for 2 h. The above treated specimen were then washed
with 0.2 M PBS (3 ꢂ 15 min), dehydrated in a standard ethanol and
acetone series, and embedded in epoxy resin at 45 °C for 12 h.
Ultrathin sections (40–60 nm) were cut with a Leica Ultracut
RM2265 (Leica, Vienna, Austria), mounted on regular hexagonal
copper grids, stained with lead citrate (10 min), washed with
ddH2O (ꢂ3), stained with uranyl acetate 30 min, washed again
with ddH2O (ꢂ3), and observed with a JEOL JEM-1230 transmission
electron microscope.
4.6. Computational details for quantitative structure–activity
relationship (QSAR) study
The most stable conformation of the synthesized compounds
were obtained using Gaussian 03 quantum chemistry package,56
at the DFT level on the basis of B3LYP/6-31G(d). All the structures
of calculated compounds were visually drawn in GaussView 4.1
and the prepared computational tasks were submitted to Gaussian
03 in GaussView 4.1 as well. Afterwards, each Gaussian output file
was loaded into Ampac’s graphic user interface (AGUI) to trans-
form itself into a .OUT file compatible with CODESSA software
(2.7.15).57 The molecular descriptors of each compound were
finally calculated using CODESSA program and they were directly
used for generation of QSAR model. Meanwhile, the lipophilicity
descriptor, an external parameter denoting octanol-water partition
coefficient (LogP) was calculated by ALOGPS 2.1 applet.58 Each
antifungal activity data was transformed into its common loga-
rithm form pEC50 (pEC50 = ꢀlogEC50). In order to explore the best
correlated models from the antifungal activity (pEC50) and the cal-
culated molecular descriptors, the heuristic method in CODESSA
was used to preselect the descriptors. The squared correction coef-
ficient (R2), the standard deviation of the regression (s2), and the
Fisher criteria (F) were chosen as the statistical parameters to indi-
cate stability and robustness of the models.
One of the most commonly used internal validation method is
the leave-one-out cross-validation. For this method, one com-
pound was excluded from the training set and the model is para-
metrized for n ꢀ 1 compounds. Then the residual value (the
difference between the observed and predicted values) was calcu-
lated for the excluded compound. Afterward, the above excluded
compound was included in the training set and another compound
was excluded. The procedure was repeated until the residual val-
ues of all the compounds were obtained. QCV2 is defined as Eq. (1).
4.8. Intracellular ROS and mitochondrial membrane potential
detections
After addition of compound 7k to the PDB culture (Section 4.7)
for 12 h, intracellular ROS in A. solani Sorauer were detected. Sam-
ples of mycelium were collected from the tip of growing mycelium
pellets and mixed with carboxy-20, 70-dichloro-dihydro-fluorescein
diacetate (DCFH-DA, Molecular Probes) solutions (10 lM) for 20
min. At the end of this period, the treated mycelium were washed
with ddH2O (ꢂ3) and fluorescence was captured at 480 nm (exci-
tation) and 527 nm (emission) wavelengths under an inverted flu-
orescent microscope (Leica DM5000B).
To monitor the change in mitochondrial transmembrane poten-
tial (DWm) in A. solani Sorauer, 12 h-old mycelium were collected
from the tip of growing mycelium pellets in PDB culture and they
were incubated in 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimi-
dazolcarbocyanine iodide (JC-1, Invitrogen) solution for 10 min in
the dark. After being washed with ddH2O, the treated mycelium
were observed under an inverted fluorescent microscope (Leica
DM5000B) with two different filter devices: green fluorescence
(excitation 470 nm, dichroic filter 495 nm, emission 525 nm) and
red fluorescence (excitation 550 12.5 nm, dichroic filter 570 nm,
emission 605 35 nm). Images with same settings were loaded
in the software Image J to quantify pixel numbers corresponding
to green and red, and then the green/red fluorescence ratios were
calculated.
ꢀ
ꢁ
2
P
n
i¼1
yoi bs ꢀ ypi red
Q2CV ¼ 1 ꢀ
ð1Þ
ꢂ
ꢃ
P
2
n
i¼1
yoi bs ꢀ y
ꢀobs
Acknowledgements
where yoi bs is the experimental (observed) value of the property for
the ith compound; ypi red is the predicted value for the temporary
This work was financially supported by National Natural
Science Foundation of China (Nos. 31272074 and 31471800). The
authors were thankful to Zhang Hongli for NMR experiments and
Tong Xiaogang for mass spectrometry.
ꢀobs
excluded ith compound; y
is the mean experimental value of
the property in the training set; and n is the total number of the
compounds in the training set.
A. Supplementary data
4.7. Ultrastructural transmission electron microscope (TEM)
observation
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2017.11.003.
In order to evaluate the effect of the most potent compound 7k
on subcellular structures in A. solani Sorauer, ultrastructural imag-
ing was performed using TEM. Firstly, a part of the fungal colony
was transferred into the potato dextrose broth (PDB, same compo-
sition as PDA but without agar) by aseptically punching out 5 mm
of the PDA plate culture with a cutter. Then, the fungal mycelium
was incubated at 25 °C for 12 h using shaker-incubator at 25
shake/min. Afterward, a certain amount of compound 7k in DMSO
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Please cite this article in press as: Delong W., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.003