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

Bioorganic & Medicinal Chemistry 25 (2017) 6661–6673

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Design, synthesis and evaluation of 3-arylidene azetidin-2-ones as potential antifungal agents against Alternaria solani Sorauer Wang Delong a, Wu Yongling a, Wang Lanying a,b, Feng Juntao a,⇑, Zhang Xing a 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

Article history: Received 21 August 2017 Revised 26 October 2017 Accepted 2 November 2017 Available online 4 November 2017 Keywords: 3-Arylidene azetidin-2-ones Antifungal activity QSAR model Subcellular effect

a b s t r a c t A new concise and facile method was explored to synthesize a collection of new 3-arylidene azetidin-2ones, 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, 13 C 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 transmembrane 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. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction 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 conservation1–4; 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 provided 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 ⇑ Corresponding author. E-mail address: [email protected] (F. Juntao). https://doi.org/10.1016/j.bmc.2017.11.003 0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.

chances for the development of resistance.9 Accordingly, there is a wake-up call currently arousing a regenerated interest in discovery 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 workshop. 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 profound 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 activities, such as antioxidant,18 antimicrobial,19 larvicidal,20,21 nematicidal, 21antiinflammatory22 and animal repellent23,24 activities. On

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involved in a SN20 attack on allyl bromides to give kinetic products,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 (tBuOK) 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-methyleneb-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 conveniently possible to determine the reaction from the 1H NMR spectra 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 deshielding 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 deshielding center of carbonyl group in the cyclic conformation (Supplementary Materials, Fig. S1). 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 different N-nucleophiles (Scheme 3). The following cyclization reactions proceeded smoothly and efficiently except that the tert-butyl precursor 6d didn’t react at all. All the NMR spectra of new compounds were included in Supplementary Materials.

the other hand, ring systems, particular heterocycles, are fundamental 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 scaffold 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 chymase 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 cinnamamide (or its derivatives) and azetidin-2-one. Their antifungal activity was screened and a quantitative structure-activity relationship (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 microscope (TEM) and fluorography techniques. These results may lay the foundation for the exploitation of this hybrid scaffold to seek potential crop-protection agents. 2. Results and discussion 2.1. Synthesis methods The synthetic route begins with Baylis-Hillman reaction of aldehydes 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 4nitrobenzaldehyde (1o) and 2-naphthaldehyde (1p) whose reactions 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 substituents (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

2.2. Antifungal activity and QSAR study of the synthesized compounds The lead compound 5a was primarily selected to test its antifungal 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 CHO R

DABCO R

neat or THF, rt, 7d 1a-p

O O

HBr/H 2SO 4

O

R

DCM, 0 oC to rt, overnight

Br

2a-p

3a-p

O NH2 R K2CO3 , CH3 CN, rt, 1h

O O

NH

3

Conditions

1

R

N

4

5a-p 4a-p Scheme 1. The synthetic routine to the target compounds 5a–p.

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Table 2 The mycelial growth inhibition rates of compound 5a against eight phytopathogenic fungi at 50.0 lg/mL.

O 1. NaOH, EtOH/H2 O

OH +

2. HCl/H2O

Cl-

NH 2•

MsCl, Bu4N+HSO4KHCO3 CHCl3 /H2 O

the reported method O R

O present study

O

R

N

NH 5a R= H; 5o R= 4-NO 2 a

4a R= H; 4o R= 4-NO2 Scheme 2. The previously reported method for synthesis of compound 5a and 5o.

Table 1 Results of different conditions for cyclization of b-aminoester 4a to a-arylidene-blactam 5a. 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 Mess 26%a,b 57%a,b 74%a,b

a On TLC plate only one product spot was visualized by UV light and Dragendoff’s reagent. b isolated yield.

Botrytis cinerea, Alternaria solani Sorauer. The results were summarized 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 investigated the influence of variations of substituents at benzene ring on the antifungal activity. As a result, the 4-Cl substitution displayed the most potent activity with EC50 of 98.2 lM (Table 3) and, accordingly, we subsequently optimized the N-substitutions with 4-Cl atom at benzene ring. Consequently, among these derivatives, compound 7k had the highest potency with EC50 of 37.6 lM, which was comparable to the commercial procymidone (30.6 lM) (Table 3). 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), hundreds of molecular descriptors of each molecule were calculated

3c

F. graminearum Sehw. G. graminis var. graminis P. oryzae Cav. S. sclerotiorum (Lib.) de Bary V. mali Miyabe et Yamada C. orbiculare B. cinerea A. solani Sorauer

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

Data are expressed as the means ± SD (n = 3).

O O

Br

Mycelial growth inhibition rate (%)a

by Codessa including constitutional, topological, geometrical, electrostatic, quantum chemical, and thermodynamic ones. The optimal 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 coefficients (R2) were plotted versus the number of descriptors and the regression calculation would be halted at the ‘‘breaking point” where the statistical increments of R2 (DR2) become less significant (<0.02). As illustrated in Fig. 1, the breaking point occurred at four descriptors, and the values of the four descriptors and the fourparameter model are given in Tables 3 and 4 respectively. The statistical parameters of the best QSAR model are as follows: R2 = 0.8778, F = 41.31, s2 = 0.0027. In Table 4, X and DX are 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, indicating that the predictive power for this model was retained after removing any one datum. The external Rext2 and Qext2 values were 0.8963 and 0.8931 respectively (Table S1, Supplementary Materials), which were similar to the training set (R2), thus simultaneously demonstrating the robustness of the generated model. From this best QSAR model, l, QC-min, QO-max and QN-min govern the antifungal activity. The dipole moment l is the most statistically significant descriptor. The l is responsible for the interactions of intermolecular dipole-dipole and dipole-induced dipole, which plays an important role in the interactions of drug with receptor.40,41 The negative value of coefficient for l indicated that molecules with lower l would possess higher antifungal activity (e.g. 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 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 electrostatic interactions with their binding sites.40,42 Indeed, atomic charges or local electron densities has been widely used to inter-

O

Cl

Fungi species

R'NH 2

O O

K2 CO 3, CH 3CN, Cl r.t., 1~3h

NH R'

LHMDS THF, -20 oC, 1h

N Cl

6a-m Scheme 3. The synthetic routine to the target compounds 7a–c, e–m.

7a-c,e-m

R'

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Table 3 Antifungal activity of compounds 5a–p and 7a–c, e–m against A. solani Sorauer and descriptors of the title compounds. Compd.

R

R0

l (Debye)

QC-min (a.u.)

QO-max (a.u.)

QN-min (a.u.)

EC50a (lM)

pEC50b

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 Procymidonec

H 4-F 4-Cl 4-Br 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 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 –

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 60.7 ± 6.6 37.6 ± 3.6 196.5 ± 13.0 250.7 ± 24.4 30.6 ± 4.6

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 –

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. b Values were obtained from pEC50 (M). c Commercial procymidone was used as control.

positive, implying their positive correlations with activity. However, 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 analyses give some useful hints for designing potential antifungal agents in the future.

1.0

0.8

R2

0.6 2.3. Effect of compound 7k on subcellular structures in A. solani Sorauer

0.4

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 membrane was visible and exhibited good adhesion to the outer cell wall, which was a bright fibrous layer. Regularly shaped mitochondria 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.

0.2 1

2

3

4

5

6

7

8

Number of descriptors 2

Fig. 1. R 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.

a

Descriptor No.

X

± DX

t-Test

Descriptora

0 1 2 3 4

4.7348e+00 1.7493e01 7.8452e+00 1.6488e+00 4.9969e+00

1.2322e+00 1.4449e02 1.7681e+00 5.6585e01 3.1124e+00

3.8426 12.1067 4.4371 2.9138 1.6055

Intercept

Descriptors are defined in Table 3.

l QC-min QO-max QN-min

W. Delong et al. / Bioorganic & Medicinal Chemistry 25 (2017) 6661–6673

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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, presence of autophagy-like vesicles, and reduced density of cytoplasmic matrix (Fig. 3C). When the treatment up to 48 h, the

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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 organelles discernible due to the high vacuolization. Considering the above symptom observations, it could therefore be concluded that the mitochondria were one of the primary subcellular targets of compound 7k. As is known, mitochondria are the primary intracellular site generating ATP via oxidative phosphorylation and the major source of reactive oxygen species (ROS) generation 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 (starvation) 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 aberrant hypertrophy in mitochondria after treatment with compound 7k for 12 h, indicating mitochondrial dysfunction occurred. Therefore, we corroborated this fact using a mitochondrial transmembrane potential-sensitive dye JC-1. The healthy cells have high 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 cells, the DWm becomes low and this dissipation keeps JC-1 in the monomeric form, which produces green fluorescence. As shown in Fig. 4, significant dissipation of DWm was well observed. The mean 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 consequently contributed to the autophagy-like process and other cell impairments. Besides here, many mitochondria-targeted drugs have been reportedly demonstrated to alter the mitochondriarelated cell status, including reduction in DWm and ATP production

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.

and increase in ROS accumulation.51–54 Taken together, these results laid the foundation for the design of new mitochondria-targeted fungicides based on the scaffold 3-arylidene azetidin-2-one.

3. Conclusion We have developed a new concise and facile method to synthesize a collection of new 3-arylidene azetidin-2-ones. Their structures 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 accumulation of reactive oxygen species, dissipation of mitochondrial transmembrane potential, and an autophagy-like cell death process in A. solani Sorauer. Taken together, these results laid the foun-

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.

W. Delong et al. / Bioorganic & Medicinal Chemistry 25 (2017) 6661–6673

dation for further design of improved crop-protection agents based on the scaffold 3-arylidene azetidin-2-one. 4. Experimental

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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.

4.1. General 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 moisture-free reactions were carried out under an inert nitrogen atmosphere 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 visualized 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 13 C chemical shifts were reported in ppm and they were calibrated according to the CDCl3 peaks at 7.27 ppm and 77.2 ppm, respectively. 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. General procedure for the preparation of cinnamyl bromide intermediates 3a–p55

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. 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.

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. Particularly, the reaction systems of 4-nitrobenzaldehyde and 2-naphthaldehyde 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 overnight 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  3), dried over Na2SO4, and concentrated. The desired product was obtained through column chromatography with petroleum ether/EtOAc (20/1).

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); 13 C 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.

4.2.3. (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.

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.

4.2.13. (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.

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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. 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. 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). 4.3.1. (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. 4.3.2. (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. 4.3.3. (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. 4.3.4. (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. 4.3.5. (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

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. 4.3.7. (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); 13 C 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. 4.3.8. (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. 4.3.9. (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. 4.3.10. (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. 4.3.11. (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. 4.3.12. (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.

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4.3.13. (E)-Methyl 3-(2-chlorophenyl)-2-((propylamino)methyl) acrylate (4m) 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. 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. 4.3.15. (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(overlapped, 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. 4.3.16. (E)-Methyl 3-(naphthalen-2-yl)-2-((propylamino)methyl) acrylate (4p) 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. 4.3.17. (E)-Methyl 3-(4-chlorophenyl)-2-((isopropylamino)methyl) acrylate (6a) 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. 4.3.18. (E)-Methyl 2-((butylamino)methyl)-3-(4-chlorophenyl) acrylate (6b) 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. 4.3.19. (E)-Methyl 3-(4-chlorophenyl)-2-((isobutylamino)methyl) acrylate (6c) 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. 4.3.20. (E)-Methyl 2-((tert-butylamino)methyl)-3-(4-chlorophenyl) acrylate (6d) 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.

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4.3.21. (E)-Methyl 2-((sec-butylamino)methyl)-3-(4-chlorophenyl) acrylate (6e) 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. 4.3.22. (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. 4.3.23. (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. 4.3.24. (E)-Methyl 3-(4-chlorophenyl)-2-((octylamino)methyl) acrylate (6h) 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. 4.3.25. (E)-Methyl 3-(4-chlorophenyl)-2-((phenylamino)methyl) acrylate (6i) 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. 4.3.26. (E)-Methyl 3-(4-chlorophenyl)-2-(((4-fluorophenyl)amino) methyl)acrylate (6j) 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. 4.3.27. (E)-Methyl 3-(4-chlorophenyl)-2-(((4-chlorophenyl)amino) methyl)acrylate (6k) 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. 4.3.28. (E)-Methyl 2-(((4-bromophenyl)amino)methyl)-3-(4chlorophenyl)acrylate (6l) 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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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. 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. Typical procedure for synthesis of compounds 5a–p and 7a–c, e– m 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(trimethylsilyl)amide(LHMDS) (1.0 M in THF) (1.5 eq) and the resulting mixture 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 distilled water for 3 times, dried over Na2SO4, and evaporated. The crude product was purified by column chromatography (petroleum ether/EtOAc, 10/1). 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]+). 4.4.2. (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]+). 4.4.3. (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]+). 4.4.4. (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]+). 4.4.5. (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]+). 4.4.6. (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,

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]+). 4.4.7. (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]+). 4.4.8. (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; ESIMS: m/z 257.0 ([M+H]+). 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]+). 4.4.10. (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]+). 4.4.11. (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]+). 4.4.12. (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; ESIMS: m/z 215.0 ([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]+). 4.4.14. (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,

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CDCl3): d 164.8, 137.7, 137.0, 132.9, 130.8, 129.0, 126.7, 126.1, 120.9, 48.9, 43.7, 21.2, 19.8, 11.5; ESI-MS: m/z 215.0 ([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); 13 C 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]+). 4.4.16. (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]+). 4.4.17. (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]+). 4.4.18. (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]+). 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]+). 4.4.20. (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]+). 4.4.21. (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]+).

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4.4.23. (E)-3-(4-Chlorobenzylidene)-1-octylazetidin-2-one (7h) 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]+). 4.4.24. (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]+). 4.4.25. (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(overlapped, 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]+). 4.4.26. (E)-3-(4-Chlorobenzylidene)-1-(4-chlorophenyl)azetidin-2one (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); 13C 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-2one (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); 13C 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]+). 4.4.28. (E)-1-(4-Chlorophenyl)-3-(4-methylbenzylidene)azetidin-2one (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]+). 4.5. 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. Appropriate 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 mycelium 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]  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

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their probability values, the toxicity regression curve of probability values versus corresponding logarithmic concentrations was plotted. The EC50 (50% maximal effective concentration) of each test compound was determined by the linear regression equation. 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 transform 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 (Log P) was calculated by ALOGPS 2.1 applet.58 Each antifungal activity data was transformed into its common logarithm form pEC50 (pEC50 = log EC50). In order to explore the best correlated models from the antifungal activity (pEC50) and the calculated molecular descriptors, the heuristic method in CODESSA was used to preselect the descriptors. The squared correction coefficient (R2), the standard deviation of the regression (s2), and the Fisher criteria (F) were chosen as the statistical parameters to indicate 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 compound was excluded from the training set and the model is parametrized for n  1 compounds. Then the residual value (the difference between the observed and predicted values) was calculated 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 values of all the compounds were obtained. QCV2 is defined as Eq. (1).

Q 2CV

2 Pn  obs  ypred i¼1 yi i ¼ 1  Pn   obs obs 2 y i¼1 yi

where

yobs i

ð1Þ

is the experimental (observed) value of the property for

the ith compound; ypred is the predicted value for the temporary i obs is the mean experimental value of excluded ith compound; y the property in the training set; and n is the total number of the compounds in the training set. 4.7. Ultrastructural transmission electron microscope (TEM) observation In order to evaluate the effect of the most potent compound 7k on subcellular structures in A. solani Sorauer, ultrastructural imaging was performed using TEM. Firstly, a part of the fungal colony was transferred into the potato dextrose broth (PDB, same composition 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 was added to the PDB culture to give the concentration of 56.5 lg/ mL (EC75). The same amount of DMSO was also added to the control shake flasks. After 12 h, 24 h and 48 h of incubation, samples of mycelium were collected from the tip of growing mycelium pellets of treated flasks respectively. Control samples were harvested after 48 h. TEM observation was performed according to the standard

procedures previously reported by Tripti Singh et al.59 All the harvested 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  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  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 (3), stained with uranyl acetate 30 min, washed again with ddH2O (3), and observed with a JEOL JEM-1230 transmission electron microscope. 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. Samples 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 (3) and fluorescence was captured at 480 nm (excitation) and 527 nm (emission) wavelengths under an inverted fluorescent microscope (Leica DM5000B). To monitor the change in mitochondrial transmembrane potential (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 -tetraethylbenzimidazolcarbocyanine 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. Acknowledgements 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. A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmc.2017.11.003. References 1. Anderson PK, Cunningham AA, Patel NG, et al. Emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers. Trends Ecol Evol. 2004;19:535–544. 2. Fisher MC, Henk DA, Briggs CJ, et al. Emerging fungal threats to animal, plant and ecosystem health. Nature. 2012;484:186–194. 3. Giraud T, Gladieux P, Gavrilets S. Linking the emergence of fungal plant diseases with ecological speciation. Trends Ecol Evol. 2010;25:387–395. 4. Bebber DP, Gurr SJ. Crop-destroying fungal and oomycete pathogens challenge food security. Fungal Genet Biol. 2015;74:62–64. 5. Drenth A, Guest DI. Fungal and oomycete diseases of tropical tree fruit crops. Annu Rev Phytopathol. 2016;54:373–395. 6. Hollomon DW. Fungicides for Plant Diseases. eLS. John Wiley & Sons, Ltd; 2001. 7. Ivic D. Curative and eradicative effects of fungicides. In: Carisse O, ed. Fungicides. InTech; 2010:3–22.

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