Design, synthesis and antibacterial activity of cinnamaldehyde derivatives as inhibitors of the bacterial cell division protein FtsZ

Accepted Manuscript Design, synthesis and antibacterial activity of cinnamaldehyde derivatives as inhibitors of the bacterial cell division protein FtsZ Xin Li, Juzheng Sheng, Guihua Huang, Ruixin Ma, Fengxin Yin, Di Song, Can Zhao, Shutao Ma PII:

S0223-5234(15)30010-6

DOI:

10.1016/j.ejmech.2015.04.048

Reference:

EJMECH 7863

To appear in:

European Journal of Medicinal Chemistry

Received Date: 10 August 2014 Revised Date:

22 April 2015

Accepted Date: 23 April 2015

Please cite this article as: X. Li, J. Sheng, G. Huang, R. Ma, F. Yin, D. Song, C. Zhao, S. Ma, Design, synthesis and antibacterial activity of cinnamaldehyde derivatives as inhibitors of the bacterial cell division protein FtsZ, European Journal of Medicinal Chemistry (2015), doi: 10.1016/ j.ejmech.2015.04.048. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract:

Design, synthesis and antibacterial activity of cinnamaldehyde derivatives as inhibitors of the bacterial cell division

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protein FtsZ

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Xin Li a, Juzheng Sheng b, Guihua Huang c, Ruixin Ma d, Fengxin Yin b, Di Song a, Can Zhao a, Shutao Ma a, *

The cinnamaldehyde derivatives, especially compound 10, exhibited potent FtsZ-targeted antibacterial activity with an MIC

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value of 4 µg/mL against S. aureus and S. epidermidis.

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ACCEPTED MANUSCRIPT Design, synthesis and antibacterial activity of cinnamaldehyde derivatives as inhibitors of the bacterial cell division protein FtsZ Xin Li a, Juzheng Sheng b, Guihua Huang c, Ruixin Ma d, Fengxin Yin b, Di Song a, Can Zhao a, Shutao Ma a, * a

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),

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School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan 250012, China b

Institute of Biochemical and Biotechnological Drug, Key Laboratory of Chemical Biology of Natural

Products (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan 250012, China c

Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, 44 West

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Culture Road, Jinan 250012, China

Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, P. R. China

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Running title: Cinnamaldehyde derivatives as FtsZ inhibitors

*Address correspondence to this author at the Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan 250012, China; Tel/Fax:

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+86-531-88382009; E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract: In an attempt to discover potential antibacterial agents against the increasing bacterial resistance, novel cinnamaldehyde derivatives as FtsZ inhibitors were designed, synthesized and evaluated for their antibacterial activity against nine significant pathogens using broth microdilution method, and their cell division inhibitory activity against four representative strains. In the in vitro antibacterial activity, the newly synthesized compounds generally displayed better efficacy against S. aureus ATCC25923 than the others. In particular, compounds 3, 8 and 10 exerted superior or

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comparable activity to all the reference drugs. In the cell division inhibitory activity, all the compounds showed the same trend as their in vitro antibacterial activity, exhibiting better activity against S. aureus ATCC25923 than the other strains. Additionally, compounds 3, 6, 7 and 8 displayed potent cell division inhibitory activity with an MIC value of below 1 µg/mL, over 256-fold better than all the reference drugs.

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Keywords: antibacterial activity; cell division inhibitory activity; cinnamaldehyde derivatives; design;

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FtsZ; synthesis.

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ACCEPTED MANUSCRIPT 1. Introduction The rapidly increasing bacterial resistance to conventional antibiotics has resulted in the enormous difficulty in fighting against bacterial infections [1-3]. Especially, the recently emerging New Delhi metallo-β-lactamase 1 (NDM-1) superbugs has made almost all of the first-line clinical antibiotics ineffective [4]. The weak or no activity of the originally powerful antibiotics in clinic against the resistant bacteria [5-6] has necessitated a proactive effort to discover novel antimicrobial

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pharmacophores with new mechanisms [7]. The filamentous temperature-sensitive protein Z (FtsZ) plays an important role in the bacterial cell division and is widely conserved in the bacterial kingdom as well as absent in the mitochondria of higher eukaryotes. In the process of bacterial cell division, FtsZ forms single-stranded filaments and then the highly dynamic Z-ring scaffold, followed by the recruitment of other cell division proteins. Once the recruitment is accomplished, filaments bends and

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Z ring contracts, leading to the closure of the septum and then the completement of the cell division [8,9]. As FtsZ is attractive and largely unexploited as a new target for the development of antimicrobials, it has aroused many interests among researchers for the exploration of novel

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antimicrobials against resistant bacteria in recent years [10-12].

The recently intensive investigation of FtsZ inhibitors as antibacterial agents leads to various anti-FtsZ molecules, such as cinnamaldehyde, totarol and PC190723 (Fig. 1) [8, 13-14]. Among these inhibitors, cinnamaldehyde displays broad-spectrum antibacterial activity with MIC values of 1000 and 500 µg/mL against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis), respectively [13]. In the target identification, FtsZ has been confirmed to be the target of cinnamaldehyde with the application of standard molecular biology experiments. Moreover, the STD-NMR spectroscopy [15-19] and in

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silico docking model with AutoDock software have further indicated that the binding region of cinnamaldehyde is located in the C-terminal region of FtsZ around the T7 loop [13]. Furthermore, its congener, trans-cinnamic acid with mild antibacterial activity, has also been substantiated to target FtsZ using light scattering assay. However, cinnamaldehyde is to be studied as a lead compound of FtsZ inhibitors against the growing bacterial resistance.

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As a continuous work of our previous investigation searching for novel and potent anti-FtsZ agents [20-21], we have carried out a novel program to design and synthesize a new library of

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cinnamaldehyde derivatives for screening potential FtsZ inhibitors. It is hoped that the cinnamaldehyde derivatives could exhibit more potent anti-FtsZ effect with a higher antibacterial efficacy and a broader antibacterial spectrum.



2. Chemistry In the present work, the target compounds 3-31 were synthesized from substituted benzaldehyde 1 as outlined in Scheme 1. The reaction of 1 with malonic acid gave cinnamic acid 2 in the presence of pyridine through the knoevenagel condensation. The chlorination of 2 with oxalyl chloride was followed by the amidation reaction with different amides in the presence of triethylamine to provide the target compounds 3-31. The newly synthesized compounds were characterized by MS, 1H NMR and IR, 3

ACCEPTED MANUSCRIPT and all the spectral data were in agreement with the proposed structures.



3. Antibacterial evaluation All the synthesized compounds (3-31) were determined for their biological activity, including the

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in vitro antibacterial activity and the cell division inhibitory activity. The in vitro antibacterial activity was performed with the application of the standard broth microdilution method recommended by NCCLS [22]. Cinnamic acid, curcumin and ciprofloxacin were selected as the reference agents in this test. Among these references, cinnamic acid and curcumin were used as the FtsZ-targeted references. And ciprofloxacin, as a representative fluoroquinolone antibacterial, was applied as the reference

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targeting bacterial DNA gyrase. It binded to the GyrA subunit of DNA gyrase belonging to the type II topoisomerase family, and then traped the double strand cleaved DNA−gyrase complex, leading to bacterial cell death [23]. The tested strains includes three strains of Staphylococcus aureus (S. aureus)

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ATCC25923, S. aureus ATCC29213 (MRSA) and S. aureus PR, two strains of Streptococcus pyogenes (S. pyogenes) PS and S. pyogenes PR, one strain of B. subtilis ATCC9372 and one strain of Pseudomonas aeruginosa (P. aeruginosa) ATCC27853, one clinically isolated strain of Staphylococcus epidermidis (S. epidermidis), and one strain of E. coli ATCC25922.

The cell division inhibitory activity of these compounds against B. subtilis, E. coli, P. aeruginosa and S. aureus was assessed by analyzing the cell length with the application of phase-contrast light microscopy [24]. The minimum drug concentration at which filamentation of Bacillus subtilis, E. coli

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and P. aeruginosa or ballooning of S. aureus appeared was recorded as cell division inhibitory concentration indicating their on-target activity. The results of minimum antibacterial activity and cell division inhibitory activity in the unit of µg/mL are shown in Table 1 and Table 2.



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The cinnamaldehyde derivatives were derived from cinnamaldehyde which displayed a definitely

FtsZ-targeted antibacterial activity and influenced FtsZ polymerization. To determine the effects of these newly synthetic compounds on the polymerization of FtsZ, a standard light scattering assay was carried out for three representatives. The inhibition of selected compounds on FtsZ polymerization was shown in Fig. 2.



As the polymerization and depolymerization of FtsZ depended on the hydrolysis of GTP, the production of inorganic phosphate could reflect the activity of FtsZ. To further validate the FtsZ-targeted mechanism of cinnamaldehyde derivatives, the light scattering assay was carried out for 4

ACCEPTED MANUSCRIPT three representatives. The inhibition of selected compounds on FtsZ GTPase was shown in Fig. 3.



4. Results and Discussion In the in vitro antibacterial activity, all the newly synthesized compounds displayed superior or

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comparable efficacy to the precursor cinnamic acid, indicating a successful modification in this program. Especially, the subseries of compounds containing 2-methylbenzimidazolyl moiety generally showed better efficacy than the others against all the tested strains. It was owing to the special structure of this fragment that was able to interact with receptors via hydrogen bonds and/or hydrophobic interactions. As the previous study revealed that cinnamaldehyde bound around the T7 loop of the

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C-terminal domain [13] and PC190723 bound to FtsZ in a cleft between helix seven (H7) and the C-terminal domain, it was rational to deduce that cinnamaldehyde derivatives might partially bound to the binding region of PC190723 [8]. And the 2-methylbenzimidazolyl moiety was predicted to bind

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into the hydrophobic channel formed by amino acid residues I172, E185, N188, I228 and I230, which also were the binding sites of the thiazolopyridine portion of PC190723 [8]. Additionally, the nitrogen atoms of 2-methylbenzimidazolyl moiety might form hydrogen bonds with amino acid residues R191 and Q192, which formed hydrogen bonds with the phenoxy ether of PC190723 as well [8]. Among these outstanding compounds, 3, 8 and 10 exhibited the most excellent activity with MIC values of 4, 10 and 4 µg/mL against S. aureus ATCC25923, comparable or superior to all the references. Furthermore, 4 and 10 exerted antibacterial activity with the same MIC value of 4 µg/mL against S.

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epidermidis, over 32-fold better than all the reference drugs. These outstanding compounds, except 3 having no substituent at the benzene ring, had chlorine atom(s) at the 4- or/and 2-position of the benzene ring, implying that the substituents at both positions contributed to the entire antibacterial activity. Furthermore, 6 with a fluorine atom at the 4-position has the comparable activity to its 4-chlorine congener 8 against all the tested strains. However, 4-methoxy derivative 9 showed a

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decreased activity against most pathogens tested. All the results above indicated that the small group at the 4- or/and 2-position of the benzene ring might increase the entire activity against various

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pathogens. This might be due to the binding region of cinnamaldehyde derivatives that was volume-limited and had the existing amino acid residues L209, L200, N208 and G205 around the T7 loop forming hydrophobic interactions with the 2,4-disubstituted benzene ring [13]. In the other subseries, the amides with two substituents generally displayed better efficacy than those with one substituent, revealing hydrophobic interaction existing between the ligand and the receptor around the amide group or no hydrogen bond donator at the receptor near the binding site. In this subseries, 20, 28, 29 and 30 exhibited better antibacterial activity against S. aureus ATCC25923 and S. aureus PR than the others. For instance, 20 and 28 exerted the same efficacy against S. aureus ATCC25923 and S. aureus PR with MIC values of 32 and 64 µg/mL, respectively. In the cell division inhibitory assay, most of the tested compounds showed more potency against S. aureus ATCC25923 than the other tested strains. And 2-methylbenzimidazolyl derivatives displayed more advantages in general than the others in the inhibition of the tested pathogens, exerting similar 5

ACCEPTED MANUSCRIPT trends to their in vitro antibacterial activity. Among these derivatives, 3, 6, 7 and 8 exhibited the most potent activity with MIC values of 0.25, 0.50, 0.50, and 0.50 µg/mL, respectively, similar to the previously discovered most potent FtsZ inhibitors [8]. In the other subseries, 16, 20, 25, 26, 28, 29 and 30 exerted the apparent inhibition of bacterial cell division with MIC values of below 128 µg/mL. All the results described above revealed that the amides with hydrophobic bulky group might increase their cell division inhibitory activity and the basic substituents at the amide nitrogen probably contributed to

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the entire cell division inhibition. In the light scattering assay, all the tested compounds displayed an obvious inhibition on FtsZ polymerization in a dose-dependent manner (Fig. 2). Among them, compound 10 displayed the best activity in the various concentration range, and it exhibited significant activity even at the concentration of 30 µg/mL, similar to compound 6 at the concentration of 120 µg/mL. The results

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indicated that these compounds inhibited the proliferation of bacteria via influencing the function of FtsZ.

In the GTPase assay, all the tested compounds showed an apparent effect on the GTPase activity

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of FtsZ and the effect was dose-dependent. Especially, compound 10 showed an inhibition of 50% at the concentration of 30 µg/mL, superior to the other compounds. The inhibition of GTPase activity resulted in the instability of the FtsZ polymer, leading to the abnormal bacterial cell division and then the cell death.

In this program, all the active compounds are promising, representing a new array of molecules with novel scaffold targeting FtsZ. The further biological investigation of these compounds is underway.

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5. Conclusions

In conclusion, a novel library of cinnamaldehyde derivatives were designed, synthesized and tested for their in vitro antibacterial activity and cell inhibitory activity against various pathogen strains, including Gram-positive and -negative bacteria. In addition, the light scattering assay and GTPase assay were carried out for the further validation of the FtsZ-targeted mechanism. Generally, the

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cinnamaldehyde derivative 3-12, bearing 2-methylbenzimidazolyl fragment, exhibited better activity than the others against all the strains, and many compounds in this subseries showed superior or

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comparable antibacterial activity to the references. Moreover, these active compounds commonly displayed better activity against S. aureus ATCC25923 than the other strains. In addition, almost all the compounds exhibited similar in vitro antibacterial activity against B. subtilis ATCC 9372, E. coli ATCC 25922 and P. aeruginosa ATCC 27853, suggesting little or no effects of different amide fragments of these molecules on the antibacterial activity. In cell division inhibitory activity, the compounds showed preferable activity against the S. aureus ATCC25923to the other strains with the minimum cell division inhibitory activity of 0.25 µg/mL, over 512-fold better than all the references. Moreover, three representatives were selected for the further evaluation using light scattering assay and GTPase assay, and the results strongly validated the mechanism of these compounds. These results indicated that the subseries were promising and worth of further investigation for potent FtsZ-targeted antibacterial agents especially against S. aureus ATCC25923. 6. Experimental Protocol 6

ACCEPTED MANUSCRIPT 6.1. Chemistry All necessary reagents and chemicals used in the present study were of analytical grade. Reactions progress was monitored by thin-layer chromatography (TLC) on 0.25-mm pre-coated silica GF254 plates (Qingdong Yumingyuan silica gel reagent factory, Shandong, China, YUYUAN). Flash column chromatography was carried out with the indicated solvents using silica gel 60 (particle size 0.040-0.063 mm, Qingdong Yumingyuan silica gel reagent factory, Shandong, China, YUYUAN). Mass spectra were obtained on the API 4000 instrument (Applied Biosystems, Connecticut, USA) 1H

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nuclear magnetic resonance (1H NMR) spectra were recorded on the Bruker Avance DRX 400 spectrometer (Bruker, Switzerlands) using appropriate deuterated solvents and were expressed in parts per million (δ, ppm) downfield from tetramethylsilane (internal standard). Infrared (IR) spectra were recorded on the Nicolet Nexus 470FT-IR spectrometer (Wisconsin, USA) using KBr pellets. Melting

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points were uncorrected and determined with the X-6 melting point apparatus (Beijing Tianchengwode Biotech Co. Ltd, Beijing, China). Bacterial morphometric analysis was determined on the Olympus CKX41 microscope.

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6.1.1. Substituted cinnamic acids (2)

To a solution of the substituted benzaldehyde (50 mmol) and malonic acid (150 mmol) in DMF (30 mL) was added pyridine (50 mmol) and stirred for 3-5 h at 90°C. After adding water (60 mL), the reaction solution was acidified (pH 1) with concentrated hydrochloric acid and then was cooled to 0°C. Then the residue was filtered, washed with cold water (2×10 mL) and dried in vacuum for 12 h to afford corresponding crude product 2 in yields ranging from 30.2 to 100.0%, which was used directly without further purification.

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6.1.2. General procedure for cinnamaldehyde derivatives (3-31)

To a solution of intermediate 2 (1.35 mmol) in THF (10 mL) at 0°C was slowly added oxalyl chloride (4.05 mmol). The reaction solution was stirred for 15-25 min at the same temperature and concentrated in vacuo to give substituted cinnamoyl chloride. The resulting crude product and TEA (4.05 mmol) was dissolved in DMF (5 mL) in an ice bath, and the corresponding amine (2.03 mmol) in

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DMF (2 mL) was dropwise added. The resulting mixture was slowly warmed to room temperature and stirred for 18 h at the same temperature. After adding water (20 mL), the compounds 3-31 were

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extracted with ethyl acetate (3×20 mL), and then the combined organic layers were was washed with saturated NaHCO3 solution (2×10 mL) and brine (2×10 mL), respectively. The washed organic layer was dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure to dryness. The residue was purified by flash chromatography (dichloromethane/methanol, 30:1 or petroleum ether/ethyl acetate, 1:1) to give compounds 3-39 in yields ranging from 42.9–91.7%. 6.1.2.1. (E)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)-3-phenylprop-2-en-1-one (3) White solid, yield 42.9%, mp 95–98°C, Rf = 0.62 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.94 (d, 1H, J = 15.6 Hz), 7.89-7.87 (m, 2H), 7.79-7.77 (m, 1H), 7.66-7.64 (m, 1H), 7.52-7.47 (m, 4H), 7.36-7.30 (m, 2H), 2.79 (s, 3H); IR (KBr): 3373, 3056, 2973, 2927, 1698, 1619, 1576, 1546, 1495, 1455, 1424, 1384, 1350, 1333, 1310, 1292, 1269, 1238, 1206, 1167, 1112, 1091, 1036 cm-1; HRMS (ESI) m/z calcd. for C17H14N2O 263.1179 [M+H]+, found: 263.1184. 7

ACCEPTED MANUSCRIPT 6.1.2.2. (E)-3-(2-chlorophenyl)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)prop-2-en-1-one (4) Light yellow solid, yield 63.6%, mp 106–108°C, Rf = 0.61 (dichloromethane/methanol, 10:1); IR (KBr): 3384, 3060, 2972, 2926, 1701, 1621, 1545, 1457, 1379, 1332, 1302, 1286, 1238, 1172, 1130, 1113, 1052, 1035 cm-1; 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.16 (d, 1H, J = 15.6), 8.11 (dd, 1H, J = 1.6, J = 2.0), 7.82-7.80 (m, 1H), 7.66-7.63 (m, 1H), 7.61-7.60 (m, 1H), 7.55-7.50 (m, 2H), 7.48 (dd, 1H, J = 0.8, J = 0.8), 7.36-7.33 (m, 2H), 2.80 (s, 3H); MS (ESI) m/z calcd for C17H13ClN2O 296.07; found

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[M+H]+ 297.5. 6.1.2.3. (E)-3-(2-methoxyphenyl)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)prop-2-en-1-one (5)

White solid, yield 75.8%, mp 87–89°C, Rf = 0.61 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.12 (d, 1H, J = 16 Hz), 7.85-7.83 (m, 1H), 7.81-7.79 (m, 1H), 7.66-7.64 (m, 1H), 7.55-7.48 (m, 2H), 7.36-7.30 (m, 2H), 7.17 (d, 1H, J = 8.4 Hz), 7.07 (t, 1H, J = 7.4 Hz), 3.93 (s, 3H), 2.78 (s, 3H); IR (KBr): 3053, 3019, 2951, 2840, 1685, 1619, 1611, 1572, 1549, 1489,

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1455, 1432, 1380, 1348, 1339, 1308, 1279, 1247, 1236, 1167, 1124, 1113, 1051, 1037, 1019 cm-1; HRMS (ESI) m/z calcd. for C18H16N2O2 293.1285 [M+H]+, found: 293.1289.

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6.1.2.4. (E)-3-(4-fluorophenyl)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)prop-2-en-1-one (6) White solid, yield 67.6%, mp 138–140°C, Rf = 0.59 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.99-7.93 (m, 3H), 7.79-7.77 (m, 1H), 7.66-7.64 (m, 1H), 7.45 (d, 1H, J = 15.6 Hz), 7.37-7.32 (m, 4H), 2.79 (s, 3H); IR (KBr): 3069, 2937, 1698, 1620, 1598, 1546, 1508, 1456, 1418, 1380, 1350, 1339, 1310, 1296, 1271, 1229, 1198, 1161, 1115, 1037, 1001 cm-1; HRMS (ESI) m/z calcd. for C17H13FN2O 281.1085 [M+H]+, found: 281.1088.

6.1.2.5. (E)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)-3-(4-nitrophenyl)prop-2-en-1-one (7)

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Brown solid, yield 90.6%, mp 178–181°C, Rf = 0.60 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.32 (d, 2H, J = 8.8 Hz), 8.16 (d, 2H, J = 8.8 Hz), 8.02 (d, 1H, J = 16 Hz), 7.82-7.79 (m, 1H), 7.70 (d, 1H, J = 16 Hz), 7.67-7.65 (m, 1H), 7.36-7.33 (m, 2H), 2.80 (s, 3H); IR (KBr): 3380, 3080, 2973, 2926, 1700, 1622, 1600, 1549, 1514, 1455, 1428, 1341, 1309, 1291, 1268,

308.1035.

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1237, 1163, 1114, 1034 cm-1; HRMS (ESI) m/z calcd. for C17H13N3O3 308.1030 [M+H]+, found: 6.1.2.6. (E)-3-(4-chlorophenyl)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)prop-2-en-1-one (8)

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White solid, yield 60.6%, mp 94–97°C, Rf = 0.55 (dichloromethane/methanol, 10:1); IR (KBr): 3473, 3369, 3081, 2972, 2927, 1695, 1624, 1591, 1568, 1539, 1491, 1454, 1436, 1408, 1382, 1346, 1314, 1286, 1266, 1240, 1208, 1155, 1104, 1092, 1063, 1012 cm-1; 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.95-7.91 (m, 3H), 7.79-7.77 (m, 1H), 7.66-7.64 (m, 1H), 7.58 (d, 2H, J = 2.4), 7.50 (d, 1H, J = 15.6), 7.36-7.31 (m, 2H), 2.79 (s, 3H); MS (ESI) m/z calcd for C17H13ClN2O 296.07; found [M+H]+ 297.5.

6.1.2.7. (E)-3-(4-methoxyphenyl)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)prop-2-en-1-one (9) White solid, yield 81.8%, mp 83–86°C, Rf = 0.57 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.92 (d, 1H, J = 15.6 Hz), 7.86 (d, 2H, J = 8.8 Hz), 7.77-7.74 (m, 1H), 7.65-7.63 (m, 1H), 7.35-7.30 (m, 3H), 7.06 (d, 2H, J = 8.8 Hz) 3.84 (s, 3H), 2.78 (s, 3H); IR (KBr): 3012, 2973, 2931, 2839, 1691, 1600, 1570, 1541, 1514, 1453, 1426, 1378, 1357, 1313, 1298, 1255, 1166, 1109, 1038, 1027, 1008 cm-1; HRMS (ESI) m/z calcd. for C18H16N2O2 293.1285 [M+H]+, found: 8

ACCEPTED MANUSCRIPT 293.1286. 6.1.2.8. (E)-3-(2,4-dichlorophenyl)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)prop-2-en-1-one (10) Light yellow solid, yield 83.3 %, mp 108–111°C, Rf = 0.61 (dichloromethane/methanol, 10:1); IR (KBr): 3058, 2925, 1698, 1617, 1609, 1582, 1550, 1469, 1456, 1387, 1352, 1335, 1308, 1294, 1275, 1237, 1168, 1113, 1099, 1052, 1035 cm-1; 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.15-8.06 (m, 2H), 7.81-7.79 (m, 2H), 7.66-7.63 (m, 1H), 7.61-7.56 (m, 2H), 7.35-7.32 (m, 2H), 2.80 (s, 3H); MS (ESI)

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m/z calcd for C17H12Cl2N2O 330.03; found [M+H]+ 331.3. 6.1.2.9. (E)-3-(3,4-dimethoxyphenyl)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)prop-2-en-1-one (11)

Yellow solid, yield 80.6%, mp 135–138°C, Rf = 0.53 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.90 (d, 1H, J = 15.6 Hz), 7.76-7.74 (m, 1H), 7.66-7.64 (m, 1H), 7.51 (d, 1H, J = 1.6 Hz), 7.45 (dd, 1H, J = 1.6 Hz, J = 1.6 Hz), 7.38 (d, 1H, J = 15.6 Hz), 7.35-7.30 (m, 2H),

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7.07 (d, 1H, J = 8.4 Hz), 3.84 (d, 6H, J = 1.2 Hz), 2.78 (s, 3H); IR (KBr): 3073, 3054, 2993, 2937, 2839, 1683, 1609, 1593, 1580, 1515, 1455, 1425, 1382, 1340, 1307, 1274, 1254, 1235, 1174, 1159, 1138, 1114, 1035, 1024 cm-1; HRMS (ESI) m/z calcd. for C19H18N2O3 323.1390 [M+H]+, found:

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323.1391.

6.1.2.10. (E)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (12) White solid, yield 66.7%, mp 172–175°C, Rf = 0.61 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.88 (d, 1H, J = 15.6 Hz), 7.76-7.74 (m, 1H), 7.66-7.64 (m, 1H), 7.49 (d, 1H, J = 15.6 Hz), 7.34-7.31 (m, 2H), 7.25 (s, 2H), 3.85 (s, 6H), 3.74 (s, 3H), 2.79 (s, 3H); IR (KBr): 2971, 2937, 2841, 2823, 1700, 1624, 1579, 1542, 1504, 1455, 1421, 1378, 1324, 1298, 1269, 1241, 1169, 1126, 1111, 1035, 1004 cm-1; HRMS (ESI) m/z calcd. for C20H20N2O4 353.1496 [M+H]+, found:

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353.1498.

6.1.2.11. N-(4-methoxyphenyl)cinnamamide (13)

Brown solid, yield 64.7%, mp 150–153°C, Rf = 0.83 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 10.10 (s, 1H), 7.64-7.55 (m, 5H), 7.47-7.40 (m, 3H), 6.93-6.91 (m, 2H), 6.82 (d, 1H, J = 15.6 Hz), 3.74 (s, 3H); IR (KBr): 3248, 3126, 3060, 2931, 2832, 1654, 1619, 1600,

EP

1540, 1509, 1464, 1449, 1412, 1344, 1297, 1239, 1178, 1108, 1038 cm-1; HRMS (ESI) m/z calcd. for C16H15NO2 254.1176 [M+H]+, found: 254.1177.

AC C

6.1.2.12. (E)-N-butyl-3-(4-cyanophenyl)acrylamide (14) White solid, yield 80.8%, mp 98–100°C, Rf = 0.60 (dichloromethane/methanol, 10:1); 1H NMR

(400 MHz, DMSO-d6, δ ppm) 8.21 (t, 1H, J = 5.4 Hz), 7.88 (d, 2H, J = 8.4 Hz), 7.75 (d, 2H, J = 8.4 Hz), 7.48 (d, 1H, J = 16 Hz), 6.77 (d, 1H, J = 16 Hz), 3.22-3.17 (m, 2H), 1.49-1.42 (m, 2H), 1.37-1.29 (m, 2H), 0.90 (t, 3H, J = 7.4 Hz); IR (KBr): 3265, 3069, 2971, 2924, 2861, 2228, 1657, 1621, 1552, 1508, 1466, 1431, 1411, 1336, 1308, 1281, 1229, 1158, 1119 cm-1; HRMS (ESI) m/z calcd. for C14H16N2O 229.1335 [M+H]+, found: 229.1332. 6.1.2.13. (E)-N-butyl-3-(4-nitrophenyl)acrylamide (15) Yellow solid, yield 57.7%, mp 121–124°C, Rf = 0.62 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.27-8.22 (m, 3H), 7.83 (d, 2H, J = 8.8 Hz), 7.52 (d, 1H, J = 16 Hz), 6.82 (d, 1H, J = 16 Hz), 3.22-3.17 (m, 2H), 1.48-1.42 (m, 2H), 1.35-1.30 (m, 2H), 0.92-0.87 (m, 3H); IR (KBr): 3305, 3064, 2956, 2934, 2873, 1654, 1615, 1594, 1549, 1515, 1473, 1455, 1411, 1342, 1221, 9

ACCEPTED MANUSCRIPT 1108, 1022 cm-1; HRMS (ESI) m/z calcd. for C13H16N2O3 249.1234[M+H]+, found: 249.1231. 6.1.2.14. (E)-N-(3-(2-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-3-(4-nitrophenyl)acrylamide (16) Yellow solid, yield 44.2%, mp 167–170°C, Rf = 0.47 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 10.87(s, 1H), 8.29 (d, 1H, J = 8.8 Hz), 8.21 (d, 1H, J = 1.2 Hz), 8.11-8.08 (m, 1H), 8.02 (s, 1H), 7.92-7.90 (m, 1H), 7.78-7.71 (m, 1H), 7.42 (d, 1H, J = 40.4 Hz),

RI PT

7.00-6.94 (m, 2H), 6.82 (s, 1H), 5.83 (d, 1H, J = 47.6 Hz), 2.19-2.16 (m, 3H); IR (KBr): 3354, 3099, 2973, 2927, 1687, 1638, 1621, 1577, 1519, 1496, 1449, 1405, 1383, 1339, 1317, 1258, 1239, 1203, 1165, 1127, 1111, 1080, 1049, 1010 cm-1; HRMS (ESI) m/z calcd. for C20H15F3N4O3 417.1169 [M+H]+, found: 417.1171. 6.1.2.15. (E)-3-(4-nitrophenyl)-N-propylacrylamide (17)

SC

Yellow solid, yield 91.7%, mp 125–128°C, Rf = 0.56 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.27-8.25 (m, 3H), 7.84-7.82 (d, 2H, J = 8.8 Hz), 7.55-7.51 (d, 1H, J = 16 Hz), 6.83 (d, 1H, J = 16 Hz), 3.19-3.14 (m, 2H), 1.54-1.45 (m, 2H), 0.92-0.86 (m, 3H); IR (KBr):

M AN U

3299, 3257, 3077, 2964, 2923, 2875, 1655, 1623, 1594, 1555, 1516, 1462, 1428, 1413, 1348, 1337, 1286, 1250, 1226, 1182, 1156, 1109 cm-1; HRMS (ESI) m/z calcd. for C12H14N2O3 235.1077 [M+H]+, found: 235.1075.

6.1.2.16. (E)-3-(4-nitrophenyl)-N-(prop-2-ynyl)acrylamide (18)

White solid, yield 61.5%, mp 234–237°C, Rf = 0.67 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.72 (t, 1H, J = 5.6 Hz), 8.27 (d, 2H, J = 8.8 Hz), 7.85 (d, 2H, J = 8.8 Hz), 7.58 (d, 1H, J = 16 Hz), 6.82 (d, 1H, J = 16 Hz), 4.03-4.00 (m, 2H), 3.19 (t, 1H, J = 2.4 Hz); IR

TE D

(KBr): 3258, 3077, 2961, 2927, 2851, 1662, 1625, 1600, 1558, 1513, 1420, 1346, 1331, 1261, 1226, 1183, 1110, 1041 cm-1; HRMS (ESI) m/z calcd. for C12H10N2O3 231.0764 [M+H]+, found: 231.0766. 6.1.2.17. (E)-N-isopropyl-3-(4-nitrophenyl)acrylamide (19) White solid, yield 66.7%, mp 165–167°C, Rf = 0.48 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.26 (d, 2H, J = 8.8 Hz), 8.16 (d, 1H, J = 7.6 Hz), 7.82 (d, 2H, J = 8.8

EP

Hz), 7.52 (d, 1H, J = 16 Hz), 6.79 (d, 1H, J = 15.6), 4.00-3.93 (m, 1H), 1.13 (d, 6H, J = 6.8 Hz); IR (KBr): 3303, 3078, 2974, 2925, 2850, 1655, 1618, 1598, 1543, 1515, 1467, 1407, 1346, 1277, 1224,

AC C

1173, 1130, 1109, 1004 cm-1; HRMS (ESI) m/z calcd. for C12H14N2O3 235.1077 [M+H]+, found: 235.1076.

6.1.2.18. (E)-3-(4-methoxyphenyl)-N-(pyridin-3-yl)acrylamide (20) White solid, yield 67.9%, mp 150–154°C, Rf = 0.50 (dichloromethane/methanol, 10:1); 1H NMR

(400 MHz, DMSO-d6, δ ppm) 10.35 (s, 1H), 8.84 (d, 1H, J = 2.4 Hz), 8.29-8.27 (m, 1H), 8.17-8.14 (m, 1H), 7.61-7.57 (m, 3H), 7.37 (dd, 1H, J = 4.8 Hz, J = 4.8 Hz), 7.02 (d, 2H, J = 8.8 Hz), 6.70 (d, 1H, J = 15.6 Hz), 3.81 (s, 3H); IR (KBr): 3242, 3184, 3119, 3059, 3000, 1682, 1622, 1604, 1589, 1553, 1513, 1477, 1427, 1346, 1304, 1287, 1251, 1194, 1169, 1129, 1113, 1025 cm-1; HRMS (ESI) m/z calcd. for C15H14N2O2 255.1128 [M+H]+, found: 255.1128. 6.1.2.19. (E)-3-(2,4-dichlorophenyl)-N-propylacrylamide (21) White solid, 88.1%, mp 144–146°C, Rf = 0.12 (petroleum ether/ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.23 (s, 1H), 7.72 (s, 2H), 7.66-7.62 (d, 1H, J = 15.6 Hz), 7.51-7.49 (d, 1H, J 10

ACCEPTED MANUSCRIPT = 8 Hz), 6.71-6.67 (d, 1H, J = 11.6 Hz), 3.15-3.14 (d, 2H, J = 5.6 Hz), 1.50-1.45 (q, 2H, J = 7.2 Hz), 0.90-0.86 (t, 3H, J = 7.2 Hz); MS (ESI) m/z calcd for C12H13Cl2NO 257.04; found [M+H]+ 258.3. 6.1.2.20. (E)-3-(2,4-dichlorophenyl)-N-isopropylacrylamide (22) White solid, 89.4%, mp 178–180°C, Rf = 0.18 (petroleum ether/ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.14-8.12 (d, 1H, J = 6.8 Hz), 7.72-7.61 (m, 3H), 7.52-7.50 (d, 1H, J = 8Hz), 6.68-6.64 (d, 1H, J = 15.6 Hz) 3.98-3.95 (m, 1H), 1.12-1.10 (d, 6H, J = 6.4 Hz); MS (ESI) m/z calcd

RI PT

for C12H13Cl2NO 257.04; found [M+H]+ 258.2. 6.1.2.21. (E)-N-butyl-3-(2,4-dichlorophenyl)acrylamide (23)

White solid, 89.2%, mp 146–149°C, Rf = 0.40 (petroleum ether/ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.21 (s, 1H), 7.72 (s, 2H), 7.66-7.62 (d, 1H, J = 15.6 Hz), 7.51-7.49 (d, 1H, J = 8.4 Hz), 6.70-6.66 (d, 1H, J = 15.6 Hz), 3.19-3.17 (d, 2H, J = 5.6 Hz), 1.44-1.43 (d, 2H, J = 6.4 Hz), 271.05; found [M+H]+ 272.5. 6.1.2.22. (E)-3-(2,4-dichlorophenyl)-N-hexylacrylamide (24)

SC

1.32-1.30 (d, 2H, J = 7.2 Hz), 0.91-0.87 (t, 3H, J = 7.2 Hz); MS (ESI) m/z calcd for C13H15Cl2NO

M AN U

White solid, 87.6%, mp 104–106°C, Rf = 0.63 (petroleum ether/ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.22 (s, 1H), 7.72-7.71 (m, 2H), 7.66-7.62 (d, 1H, J = 15.6 Hz), 7.51-7.49 (d, 1H, J = 8 Hz), 6.71-6.67 (d, 1H, J = 15.6 Hz), 3.18-3.17 (d, 2H, J = 5.6 Hz), 1.45 (s, 2H), 1.27 (s, 6H), 0.87 (s, 3H); MS (ESI) m/z calcd for C15H19Cl2NO 299.08; found [M+H]+ 300.5. 6.1.2.23. (E)-3-(2,4-dichlorophenyl)-N-(4-methoxyphenyl)acrylamide (25) White solid, 85.7%, mp 205–208°C, Rf = 0.43 (petroleum ether/ethyl acetate, 2:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 10.21 (s, 1H), 7.80-7.78 (d, 1H, J = 6 Hz), 7.76 (s, 2H), 7.63-7.61 (d, 2H, J =

TE D

8.4 Hz), 7.56-7.54 (d, 1H, J = 8Hz), 6.94-6.91 (d, 2H, J = 8.8 Hz), 6.89-6.85 (d, 1H, J = 16 Hz), 3.74 (s, 3H); MS (ESI) m/z calcd for C16H13Cl2NO2 321.03; found [M+H]+ 322.4. 6.1.2.24. (E)-3-(3,4-dimethoxyphenyl)-N-(prop-2-ynyl)acrylamide (26) White solid, yield 83.3%, mp 144–147°C, Rf = 0.88 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 8.43 (t, 1H, J = 5.6 Hz), 7.40 (d, 1H, J = 15.6 Hz), 7.17-7.11 (m, 2H),

EP

6.99 (d, 1H, J = 8 Hz), 6.51 (d, 1H, J = 15.6 Hz), 3.99-3.97 (m, 2H), 3.79 (d, 6H, J = 4 Hz), 3.14 (t, 1H, J = 2.6 Hz); IR (KBr): 3251, 3014, 2961, 2910, 2852, 1652, 1622, 1595, 1580, 1529, 1515, 1469, 1455,

AC C

1417, 1435, 1339, 1303, 1260, 1242, 1216, 1167, 1138, 1039, 1017 cm-1; HRMS (ESI) m/z calcd. for C14H15NO3 246.1125 [M+H]+, found: 246.1120. 6.1.2.25. (E)-N-butyl-3-(3,4-dimethoxyphenyl)acrylamide (27) White solid, yield 71.4%, mp 112–115°C, Rf = 0.50 (dichloromethane/methanol, 10:1); 1H NMR

(400 MHz, DMSO-d6, δ ppm) 7.96 (t, 1H, J = 5.4 Hz), 7.34 (d, 1H, J = 16 Hz), 7.14-7.09 (m, 2H), 6.98 (d, 1H, J = 8.4 Hz), 6.50 (d, 1H, J = 15.6 Hz), 3.79 (d, 6H, J = 5.6 Hz), 3.19-3.14 (m, 2H), 1.47-1.40 (m, 2H), 1.36-1.23 (m, 2H), 0.89 (t, 3H, J = 7.4 Hz); IR (KBr): 3292, 3007, 2961, 2934, 2872, 1652, 1614, 1579, 1549, 1513, 1462, 1437, 1419, 1363, 1320, 1263, 1213, 1192, 1160, 1142, 1037, 1026 cm-1; HRMS (ESI) m/z calcd. for C15H21NO3 264.1594 [M+H]+, found: 264.1595. 6.1.2.26. (E)-3-(4-fluorophenyl)-1-morpholinoprop-2-en-1-one (28) White solid, yield 78.6%, mp 134–137°C, Rf = 0.48 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.81-7.78 (m, 2H), 7.51 (d, 1H, J = 15.2 Hz), 7.27-7.20 (m, 3H), 11

ACCEPTED MANUSCRIPT 3.71-3.57 (m, 8H); IR (KBr): 3069, 2972, 2916, 2867, 1648, 1610, 1598, 1511, 1428, 1410, 1364, 1306, 1267, 1213, 1196, 1163, 1113, 1073, 1049 cm-1; HRMS (ESI) m/z calcd. for C13H14FNO2 236.1081 [M+H]+, found: 236.1084. 6.1.2.27. (E)-3-(2-chlorophenyl)-1-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)prop-2-en-1-one (29) Yellow solid, yield 62.3%, mp 82–84°C, Rf = 0.75 (dichloromethane/methanol, 10:1); 1H NMR

RI PT

(400 MHz, DMSO-d6, δ ppm) 7.97-7.94 (m, 1H), 7.81-7.77 (d, 1H, J = 15.2 Hz), 7.52-7.50 (m, 1H), 7.48-7.46 (d, 2H, J = 8.4 Hz), 7.43-7.41 (m, 2H), 7.40-7.36 (m, 4H), 7.32 (t, 2H, J = 7.6 Hz), 7.27 (d, 1H, J = 15.2 Hz), 7.24 (d, 1H, J = 1.2 Hz), 4.41 (s, 1H), 3.73-3.60 (m, 4H), 2.32 (s, 4H); IR (KBr): 3419, 3060, 3025, 2917, 2809, 1647, 1608, 1488, 1472, 1434, 1369, 1306, 1263, 1227, 1202, 1144, 1089, 1052, 1038 cm-1; HRMS (ESI) m/z calcd. for C26H24Cl2N2O 451.1338 [M+H]+, found: 451.1340.

SC

6.1.2.28.

(E)-1-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)-3-(2-methoxyphenyl)prop-2-en-1-one (30) White solid, yield 73.2%, mp 128–131°C, Rf = 0.59 (dichloromethane/methanol, 10:1); 1H NMR

M AN U

(400 MHz, DMSO-d6, δ ppm) 7.78-7.70 (m, 2H), 7.48-7.41 (m, 3H), 7.38-7.30 (m, 4H), 7.23-7.20 (m, 1H), 7.15 (d, 1H, J= 15.6 Hz), 7.05 (d, 1H, J = 8.4 Hz), 6.98-6.94 (m, 1H), 5.76 (s, 2H), 4.40 (s, 1H), 3.84 (s, 3H), 3.64 (d, 4H, J = 40.4 Hz), 2.42 (d, 4H, J = 78.4 Hz); IR (KBr): 3427, 3026, 2998, 2959, 2919, 2807, 2760, 1643, 1600, 1488, 1463, 1427, 1370, 1312, 1246, 1226, 1195, 1162, 1144, 1106, 1085, 1044, 1000 cm-1; HRMS (ESI) m/z calcd. for C27H27ClN2O2 447.1834 [M+H]+, found: 447.1835. 6.1.2.29. (E)-3-(2,4-dichlorophenyl)-1-morpholinoprop-2-en-1-one (31) White solid, 82.1%, mp 144–147°C, Rf = 0.13 (petroleum ether/ethyl acetate, 2:1); 1H NMR (400

TE D

MHz, DMSO-d6, δ ppm) 8.08-8.06 (d, 1H, J = 8.4 Hz), 7.79-7.75 (d, 1H, J = 15.2 Hz), 7.71 (s, 1H), 7.52-7.50 (d, 1H, J = 8Hz), 7.38-7.34 (d, 1H, J = 15.2 Hz), 3.73 (s, 2H), 3.61-3.58 (m, 6H); MS (ESI) m/z calcd for C13H13Cl2NO2 285.03; found [M+H]+ 286.3. 6.1.2.30. (E)-3-(2,4-dichlorophenyl)-N,N-diethylacrylamide (32) White solid, 87.5%, mp 95–98°C, Rf = 0.37 (petroleum ether/ethyl acetate, 2:1); 1H NMR (400

EP

MHz, DMSO-d6, δ ppm) 8.06-8.04 (d, 1H, J = 8.4), 7.77-7.71 (m, 2H), 7.50-7.48 (d, 1H, J = 8.4 Hz), 7.22-7.18 (d, 1H, J = 15.6 Hz), 3.54-3.52 (d, 2H, J = 6.8 Hz), 3.39-3.37 (d, 2H, J = 6.8 Hz), 1.17-1.13

AC C

(t, 3H, J = 6.4 Hz), 1.10-1.06 (t, 3H, J = 6.4 Hz); MS (ESI) m/z calcd for C13H15Cl2NO 271.05; found [M+H]+ 272.5. 6.1.2.31.

(E)-1-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)-3-(2,4-dichlorophenyl)prop-2-en-

1-one (33)

Light yellow solid, 89.5%, mp 73–76°C, Rf = 0.24 (petroleum ether/ethyl acetate, 2:1); 1H NMR

(400 MHz, DMSO-d6, δ ppm) 8.01-7.99 (d, 1H, J = 8.4 Hz), 7.74 (s, 1H), 7.70 (s, 1H), 7.48-7.46 (m, 3H), 7.43-7.41 (m, 4H), 7.32-7.30 (m, 3H), 7.24-7.22 (m, 1H), 4.42 (s, 1H), 3.72 (s, 2H), 3.60 (s, 2H), 2.32 (s, 4H); MS (ESI) m/z calcd for C26H23Cl3N2O 484.09; found [M+H]+ 485.5. 6.1.2.32. (E)-3-(3,4-dimethoxyphenyl)-N,N-diethylacrylamide (34) White solid, yield 56.0%, mp 88–90°C, Rf = 0.59 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.44 (d, 1H, J = 15.2 Hz), 7.31 (d, 1H, J = 2 Hz), 7.21 (dd, 1H, J = 2 Hz, J = 2 Hz), 6.98 (d, 1H, J = 4.8 Hz), 6.95 (d, 1H, J = 2.4 Hz) 3.80 (d, 6H, J = 12 Hz), 3.53 (d, 2H, J = 12

ACCEPTED MANUSCRIPT 6.8 Hz), 3.37 (d, 2H, J = 7.2 Hz), 1.15 (t, 3H, J = 6.8 Hz), 1.07 (t, 3H, J = 6.8 Hz); IR (KBr): 3389, 3075, 2964, 2930, 2839, 1639, 1593, 1512, 1478, 1459, 1423, 1378, 1363, 1305, 1261, 1228, 1161, 1138, 1079, 1037, 1018 cm-1; MS (ESI) m/z calcd. for C15H21NO3 [M+H]+, found: 264.3. 6.1.2.33. (E)-3-(3,4-dimethoxyphenyl)-1-morpholinoprop-2-en-1-one (35) White solid, yield 55.6%, mp 120–123°C, Rf = 0.45 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.47 (d, 1H, J = 15.2 Hz), 7.36 (d, 1H, J = 1.6 Hz), 7.21 (dd, 1H, J = 2

RI PT

Hz, J = 1.6 Hz), 7.11 (d, 1H, J = 15.2 Hz), 6.96 (d, 1H, J = 8.4 Hz), 3.80 (d, 6H, J = 13.2 Hz), 3.72 (s, 2H), 3.61-3.57 (m, 6H); IR (KBr): 3016, 2967, 2928, 2847, 1644, 1596, 1515, 1457, 1431, 1409, 1364, 1331, 1301, 1266, 1253, 1241, 1193, 1145, 1108, 1046, 1020 cm-1; HRMS (ESI) m/z calcd. for C15H19NO4 278.1387 [M+H]+, found: 278.1386. 6.1.2.34. (E)-3-(3,4-dimethoxyphenyl)-N,N-dimethylacrylamide (36)

SC

Yellow solid, yield 58.3%, mp 100–113°C, Rf = 0.57 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.42 (d, 1H, J = 15.2 Hz), 7.34 (d, 1H, J = 2 Hz), 7.10 (dd, 1H, J = 1.6 Hz, J = 1.6 Hz), 7.08 (d, 1H, J = 15.2 Hz), 6.96 (d, 1H, J = 8.4 Hz), 3.82-3.77 (m, 6H), 3.17 (s, 3H),

M AN U

2.93 (s, 3H); IR (KBr): 2931, 2838, 1645, 1600, 1512, 1479, 1438, 1424, 1394, 1341, 1314, 1265, 1229, 1164, 1138, 1021 cm-1; HRMS (ESI) m/z calcd. for C13H17NO3 236.1281 [M+H]+, found: 236.1279. 6.1.2.35. (E)-N,N-diethyl-3-(3,4,5-trimethoxyphenyl)acrylamide (37)

White solid, yield 56.0%, mp 128–130°C, Rf = 0.58 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.43 (d, 1H, J = 15.6 Hz), 7.03 (d, 3H, J = 15.2 Hz), 3.84 (d, 6H, J = 8 Hz), 3.68 (s, 3H), 3.54 (d, 2H, J = 6.8 Hz), 3.40-3.35 (m, 2H), 1.15 (t, 3H, J = 6.8 Hz), 1.07 (t, 3H, J = 6.8 Hz); IR (KBr): 2969, 2939, 2839, 1649, 1596, 1582, 1505, 1463, 1421, 1345, 1324, 1283, 1248, [M+H]+, found: 294.1704.

TE D

1232, 1185, 1141, 1124, 1099, 1073, 1001 cm-1; HRMS (ESI) m/z calcd. for C16H23NO4 294.1700 6.1.2.36. (E)-1-morpholino-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (38) White solid, yield 57.7%, mp 127–130°C, Rf = 0.71 (dichloromethane/methanol, 10:1); 1H NMR (400 MHz, DMSO-d6, δ ppm) 7.47 (d, 1H, J = 15.2 Hz), 7.18 (d, 1H, J = 15.2 Hz), 7.05 (s, 2H), 3.83 (s,

EP

6H), 3.74 (s, 2H), 3.68 (s, 3H), 3.59 (d, 6H, J = 19.2 Hz); IR (KBr): 3447, 2952, 2848, 1644, 1596, 1584, 1506, 1455, 1419, 1341, 1323, 1296, 1273, 1251, 1228, 1193, 1157, 1122, 1066, 1046 cm-1;

AC C

HRMS (ESI) m/z calcd. for C16H21NO5 308.1492 [M+H]+, found: 308.1497. 6.1.2.37. (E)-N,N-dimethyl-3-(3,4,5-trimethoxyphenyl)acrylamide (39) Brown solid, yield 54.2%, mp 120–123°C, Rf = 0.57 (dichloromethane/methanol, 10:1); 1H NMR

(400 MHz, DMSO-d6, δ ppm) 7.42 (d, 1H, J = 15.6 Hz), 7.15 (d, 1H, J = 15.2 Hz), 7.04 (s, 2H), 3.83 (s, 6H), 3.68 (s, 3H), 3.18 (s, 3H), 2.93 (s, 3H); IR (KBr): 3063, 2999, 2942, 2849, 1654, 1614, 1580, 1503, 1467, 1454, 1421, 1396, 1336, 1314, 1267, 1247, 1187, 1123, 1001 cm-1; HRMS (ESI) m/z calcd. for C14H19NO4 266.1387 [M+H]+, found: 266.1382. 6.2. Antibacterial evaluation 6.2.1. In vitro antibacterial assay The minimum inhibitory concentration (MIC) of the tested compounds was determined applying tube dilution method recommended by NCCLS [25]. Bacterial strains were incubated on MHA (Mueller Hinton Agar) medium at 37°C for 24 h. The bacteria solution was prepared by suspension in 13

ACCEPTED MANUSCRIPT 10 mL of sterile water for colonies from culture on MHA medium. MHB (Mueller Hinton Broth) was used for bacteria in this test. The cell density of each inoculum was adjusted in sterile water of a 0.5 McFarland standard. In this method, various concentrations of the cinnamaldehyde derivatives were prepared from 128 to 0.25 µg/mL in sterile tubes No. 1 to 10. 100 µL sterile Mueller Hinton Broth (MHB) was poured in each sterile tube followed by addition of 200 µL test compound in tube 1. Two fold serial dilutions were carried out from tube 1 to tube 10 and excess broth (100 µL) was discarded

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from the last tube No. 10. To each tube, 100 µL of standard inoculum (1.5×108 cfu/mL) was added. Cinnamic acid, curcumin and ciprofloxacin were used as controls. Turbidity was observed after incubating the inoculated tubes at 37°C for 24 hrs. The last tube with no growth of microorganism was recorded to represent the MIC value expressed in µg/mL. 6.2.2. Cell division inhibitory assay

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Cell division inhibitory activity of the tested compounds was performed as described previously [24]. Overnight cultures were grown in starvation medium supplemented with 1% hydrolyzed casein and then diluted in starvation medium supplemented with 3% hydrolyzed casein (B. subtilis) or in

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Mueller-Hinton medium (S. aureus, E. coli and P. aeruginosa) and grown at 37°C. The culture was diluted to A600 of ~0.06, and 10 µL aliquots were added to transparent 96-well microtiter plates containing dilutions of the cinnamaldehyde derivatives, cinnamic acid, curcumin and ciprofloxacin as controls in 100 µL volumes of medium. After incubation for approximately 5 h (4–5 generations) at 37°C, 20 µL culture samples were transferred to poly-L-lysine-coated slides for microscopy. Cell morphology was assessed by phase-contrast light microscopy. Lowest concentration at which filamentation of B. subtilis, E. coli and P. aeruginosa or ballooning of S. aureus was recorded

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represented the cell division inhibitory activity indicating on-target activity. 6.2.3. Expression and purification of E. coli FtsZ

FtsZ (GeneBank accession number: NC_000913) was recombinant expressed in BL21 (DE3) cells as a soluble N-His6-tagged fusion protein.

The genomic DNA isolated from E. coli ATCC 25922 was

used as the template for polymerase chain reactions (PCR). of

FtsZ

protein

was

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(ORF)

amplified

And the complete open reading frame

with

the

5’

primer

FtsZ-F

[5’-

GcatgactggtggacagcaaatgggtcgcGGATCCTTTGAACCAATGAACTTA-3’] and the 3’ primer FtsZ -R Then, the PCR product

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[5’- CggatctcagtggtggtggtggtggtgCTCGAGTTAATCGCTTACGCAGG-3’].

was ligated with corresponding predigested pET28a(+) by Gibson assembly combination [26]. The ligation products were transformed into E. coli DH5α cells. Plasmid containing FtsZ was confirmed by DNA sequencing and then transformed into E. coli BL21 (DE3). The transformed cells were grown in LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L

NaCl) supplemented with 100 µg/ml kanamycin at 37 °C. The temperature was decreased to 22 °C when the A600 reached 0.4–0.8, followed by the addition of isopropyl-1-thio-β-D-galactopyranoside (0.2 mM) to induce the expression of FtsZ proteins. After shaking for 18 h, the cells were pelleted and lysed by sonication. FtsZ proteins were then purified using Ni Sepharose 6 Fast Flow (GE healthcare life sciences, Pittsburgh, PA, USA) following the protocol provided by the manufacturer. The protein product was analyzed by 10% SDS-PAGE stained with Coomassie Blue. 6.2.4. Light scattering assay 14

ACCEPTED MANUSCRIPT The inhibition of cinnamaldehyde derivatives for E. coli FtsZ was measured using a standard light scattering assay on a Hitachi fluorescence spectrophotometer (model F-7000). The excitation and emission wavelengths were set at 400 nm and a slit width of 5 nm. The gain was set at 400 V and the cuvette chamber was maintained at 30°C by a circulating water bath. E. coli FtsZ (12 µM) was incubated in the polymerization buffer (50 mM MES, 5 mM MgCl2, 50 mM KCl, pH 6.5) containing various concentrations of a cinnamaldehyde derivative and was added to a fluorometer cuvette with a

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1-cm path length. The data were collected for 120 s to establish a baseline. Then polymerization was initiated with the addition of 2 mM GTP and determined for 2000 s. Compound stock solutions were prepared in DMSO and the percentage of DMSO was maintained at 2% for all experiments. [27, 28] 6.2.5. GTPase assay

A standard Malachite Green/ammonium molybdate assay was used to determine the production of

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inorganic phosphate during the assembly of FtsZ. E. coli FtsZ (10 µM) was incubated in polymerization buffer (50 mM MES, 5 mM MgCl2, 50 mM KCl, pH 6.5) with different concentrations of a cinnamaldehyde derivative at room temperature for 15 min. Then 50 μM GTP was added to the

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reaction mixture and incubated at 37°C for 30 min. Subsequently, Malachite Green reagent (20% v/v) was added to the reaction mixtures and the reaction mixtures were centrifuged at 13 000 rpm for 90 s to remove the protein debris. The samples (100 µL) were transferred to a 96-well plate, and the absorbance of each well was measured at 620 nm. The background absorbance was subtracted from all the readings. A phosphate standard curve was created with a Malachite Green Phosphate Assay Kit (Bioassay Systems). Compound stock solutions were prepared in DMSO and the percentage of DMSO

Acknowledgements

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was maintained at 2% for all experiments. [29]

This research was supported financially by the National Natural Science Foundation of China (20872081 and 21072114), the Natural Science Foundation of Shandong (ZR2010HM092), and

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China–Australia Centre for Health Sciences Research (CACHSR, 2014GJ06).

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Appendix A. Supplementary data

Supplementary material is available on the publishers Web site along with the published article.

References

[1] M.H. Miceli, J.A. Díaz, S.A. Lee, Lancet. Infect. Dis. 11 (2011) 142-151. [2] B. Spellberg, R. Guidos, D. Gilbert, J. Bradley, H.W. Boucher, W.M. Scheld, J.G. Bartlett, J. Edwards, Clin. Infect. Dis. 46 (2008) 155-164. [3] I.M. Gould, M.Z. David, S. Esposito, J. Garau, G. Lina, T. Mazzei, G. Peters, Int. J. Antimicrob. Agents 39 (2012) 96-104. [4] K.K. Kumarasamy, M.A. Toleman, T.R. Walsh, J. Bagaria, F. Butt, R. Balakrishnan, U. Chaudhary, M. Doumith, C.G. Giske, S. Irfan, Lancet. Infect. Dis. 10 (2010) 597-602. [5] R.H. Deurenberg, E.E. Stobberingh, Curr Mol Med. 9 (2009) 100-115. 15

ACCEPTED MANUSCRIPT [6] M.E. de Kraker, M. Wolkewitz, P.G. Davey, H. Grundmann, Antimicrob. Agents Chemother. (2011) 1598-1605. [7] Y.-T. Tan, D.J. Tillett, I.A. McKay, Mol. Med. Today. 6 (2000) 309-314. [8] D.J. Haydon, N.R. Stokes, R. Ure, G. Galbraith, J.M. Bennett, D.R. Brown, P.J. Baker, V.V. Barynin, D.W. Rice, S.E. Sedelnikova, Science 321 (2008) 1673-1675. [9] E. Bi, J. Lutkenhaus, Nature (1991) 161-164. [10] T. Matsui, J. Yamane, N. Mogi, H. Yamaguchi, H. Takemoto, M. Yao, I. Tanaka, Acta Crystallogr.

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D Biol. Crystallogr. 68 (2012) 1175-1188.

[11] N.L. Elsen, J. Lu, G. Parthasarathy, J.C. Reid, S. Sharma, S.M. Soisson, K.J. Lumb, J. Am. Chem. Soc. 134 (2012) 12342-12345.

[12] M. Kaul, A.K. Parhi, Y. Zhang, E.J. LaVoie, S. Tuske, E. Arnold, J.E. Kerrigan, D.S. Pilch, J. Med. Chem. 55 (2012) 10160-10176.

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[13] P. Domadia, S. Swarup, A. Bhunia, J. Sivaraman, D. Dasgupta, Biochem. Pharmacol. 74 (2007) 831-840.

[14] R. Jaiswal, T.K. Beuria, R. Mohan, S.K. Mahajan, D. Panda, Biochem. 46 (2007) 4211-4220. [15] Y. Chen, K. Bjornson, S.D. Redick, H.P. Erickson, Biophys. J. 88 (2005) 505-514.

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[16] M.R. Caplan, H.P. Erickson, J. Biol. Chem. 278 (2003) 13784-13788.

[17] L. Romberg, M. Simon, H.P. Erickson, J. Biol. Chem. 276 (2001) 11743-11753. [18] C. Lu, M. Reedy, H.P. Erickson, J. Bacteriol. 182 (2000) 164-170. [19] J. Mingorance, S. Rueda, P. Gómez 83-91.

Puertas, A. Valencia, M. Vicente, Mol. Microbiol. 41 (2001)

[20] S. Ma, C. Cong, X. Meng, S. Cao, H. Yang, Y. Guo, X. Lu, S. Ma, Bioorg. Med. Chem. Lett. 23 (2013) 4076-4079.

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[21] S. Ma, R. Wang, Y. Wang, J. Cao, S. Ma, Lett. Drug Des. Discov., 10 (2013) 320-326. [22] J.H. Jorgensen, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard: NCCLS document M7-A3. NCCLS: 1993. [23] Hameed P, S., Patil, V., Solapure, S., Sharma, U., Madhavapeddi, P., Raichurkar, A., Chinnapattu, M., Manjrekar, P., Shanbhag, G., Puttur, J., Shinde V., Menasinakai S., Rudrapatana S., Achar V.,

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Awasthy D., Nandishaiah R., Humnabadkar V., Ghosh A., Narayan C., Ramya V.K., Kaur P., Sharma S., Werngren J., Hoffner S., Panduga V., Kumar C.N.N., Reddy J., Kumar KN, M., Ganguly S., Bharath S., Bheemarao U., Mukherjee K., Arora U., Gaonkar S., Coulson M., Waterson D.,

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Sambandamurthy V.K., de Sousa, S. M., J. Med. Chem. 57 (2014) 4889-4905. [24] N.R. Stokes, J. Sievers, S. Barker, J.M. Bennett, D.R. Brown, I. Collins, V.M. Errington, D. Foulger, M. Hall, R. Halsey, J. Biol. Chem. 280 (2005) 39709-39715. [25] J.M. Andrews, J. Antimicrob. Chemother. 48 (2001) 5-16. [26] D.G. Gibson, L. Young, R.-Y. Chuang, J.C. Venter, C.A. Hutchison, H.O. Smith, Nat. Meth. 6 (2009) 343-345. [27] A. Mukherjee, J. Lutkenhaus, J. Bacteriol. 181 (1999) 823-832. [28] D. Awasthi, K. Kumar, S.E. Knudson, R.A. Slayden, I. Ojima, J. Med. Chem. 56 (2013) 9756-9770. [29] K. Kumar, , D. Awasthi, S.Y. Lee, I. Zanardi, B. Ruzsicska, S. Knudson, P.J. Tonge, R.A. Slayden, I. Ojima, J. Med. Chem. 54 (2010) 374-381.

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ACCEPTED MANUSCRIPT Figure captions

Fig. 1. Structures of cinnamaldehyde, totarol and PC190723.

Fig. 2. Inhibition of FtsZ polymerization by 6 (A), 8 (B) and 10 (C).

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Fig. 3. Inhibition of GTPase activity of FtsZ by cinnamaldehyde derivatives 6, 8 and 10.

Scheme 1. Synthetic route for cinnamaldehyde derivatives (3-39). Regents and Conditions: (a) malonic

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acid, pyridine, reflux for 12 h; (b) oxalyl chloride, THF, 0 °C for 20 min; (c) DMF, rt for 24 h.

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ACCEPTED MANUSCRIPT Table 1 Cinnamaldehyde derivatives with their in vitro antibacterial activity (µg/mL)

b

P. aeruginosa ATCC27853

128 128 128 128 128 128 128 128 >128 >128 128 >128 >128 128 >128 >128 >128 128 >128 >128 >128 >128 >128 >128 >128 128 >128 >128 >128 >128 >128 128 128 128 128 128 128

128 128 128 128 128 128 128 128 128 128 128 >128 >128 >128 >128 >128 >128 128 128 128 >128 >128 >128 >128 >128 128 >128 >128 128 >128 >128 128 128 128 128 128 128

128 128 128 128 128 128 128 128 128 >128 128 >128 >128 >128 >128 >128 >128 128 128 128 128 >128 >128 >128 >128 128 >128 >128 128 128 >128 >128 >128 128 128 >128 128

4 32 64 16 16 8 128 4 >128 >128 >128 >128 >128 128 >128 >128 >128 32 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 32 64 64 >128

>128 128 128 >128 >128 >128 128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 128 >128 >128 >128 >128 >128 128 >128

>128

>128

>128

>128

>128

>128

32

>128

>128

>128

>128

8

8

4

8

4

B. subtilis ATCC9372 a

Compounds

E. coli ATCC25922

S. aureus ATCC25923

S. aureus ATCC29213

d

e

S. epidermidis g

S. pyogenes PS h

S. pyogenes PR i

32 4 16 16 128 16 32 4 >128 128 >128 ND ND >128 ND ND ND 128 >128 >128 >128 >128 >128 ND ND >128 >128 >128 64 32 >128 >128 >128 >128 >128 >128 >128

128 >128 128 128 64 32 >128 >128 64 128 >128 ND ND 32 ND ND ND >128 128 64 64 128 128 ND ND >128 >128 >128 >128 128 >128 >128 >128 >128 >128 >128 >128

128 >128 >128 64 16 8 >128 64 64 128 >128 ND ND 128 ND ND ND 128 >128 >128 128 >128 128 ND ND >128 >128 >128 128 128 >128 >128 >128 >128 >128 >128 >128

>128

>128

>128

>128

ND

>128

ND

8

>128

8

>128

S. aureus PR f

c

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B. subtilis ATCC9372: penicillin-susceptible strain; b E. coli ATCC25922: penicillin-susceptible strain; P.

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64 64 64 64 128 64 64 16 >128 >128 >128 >128 >128 >128 >128 >128 >128 64 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 64 64 >128 >128

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c

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 cinnamic acid curcumin ciprofloxaci n

aeruginosa

ATCC27853:

penicillin-susceptible strain;

e

penicillin-susceptible

strain;

S.

aureus

ATCC25923:

f

S. aureus PR:

S. aureus ATCC29213: methicillin-resistant strain;

penicillin-resistant strain isolated clinically, not characterized;

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d

h

g

S. epidermidis: penicillin-resistant

strain isolated clinically, not characterized; S. pyogenes PS: penicillin-susceptible strain; i S. pyogenes PR: penicillin-resistant strain; ND: not determine.

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ACCEPTED MANUSCRIPT Table 2 Cinnamaldehyde derivatives with their cell division inhibitory activity (µg/mL)

a c

>128 128 128 >128 >128 >128 >128 >128 128 >128 128 >128 >128 >128 >128 >128 >128 128 >128 >128 >128 >128 >128 >128 >128 128 >128 >128 >128 >128 >128 128 128 128 128 128 128 >128 >128 >128

S. aureus ATCC25923 d

0.25 16 >128 0.5 0.5 0.5 >128 1 64 64 >128 >128 >128 32 >128 >128 >128 4 64 32 >128 >128 >128 >128 >128 >128 >128 64 >128 128 64 64 >128 8 32 16 >128 >128 >128 >128

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>128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 128 >128 >128 >128 >128 32 32 >128 128 >128 >128 >128 128 >128 >128 >128 >128 64 128 >128 >128 128 128 >128 >128 16 >128

P. aeruginosa ATCC27853 c >128 128 128 >128 >128 >128 >128 >128 >128 >128 128 >128 >128 128 >128 >128 >128 128 >128 >128 >128 >128 >128 >128 >128 128 128 >128 >128 >128 >128 128 >128 >128 128 >128 128 >128 ND >128

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3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 cinnamic acid curcumin ciprofloxacin

a

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B. subtilis ATCC9372

Compounds

B. subtilis ATCC9372: penicillin-susceptible strain; b E. coli ATCC25922: penicillin-susceptible strain; P.

aeruginosa

ATCC27853:

penicillin-susceptible

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penicillin-susceptible strain.

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strain;

d

S.

aureus

ATCC25923:

ACCEPTED MANUSCRIPT Fig. 1 O

NH2

F

F

OH S

O O

N N

Cl totarol

PC190723

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cinnamaldehyde

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Scheme 1.

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> Novel cinnamaldehyde derivatives as FtsZ inhibitors were designed, synthesized and evaluated. > These derivatives had excellent activity against S. aureus strains, especially S. aureus ATCC25923. > They also showed greatly improved

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activity against all the tested strains compared with their precursor. > The best compounds bearing

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2-methylbenzimidazolyl moiety had have chlorine atom(s) at the 4- or/and 2-position of the benzene ring.

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Design, synthesis and antibacterial activity of cinnamaldehyde derivatives as inhibitors of the bacterial cell division protein FtsZ

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Supplementary material

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Index: General Experimental Procedures

S2—S30

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IR, 1H NMR and MS Spectra

S1

General Experimental Procedures. All necessary reagents and chemicals used in the present study were of analytical grade. Reactions progress was monitored by thin-layer chromatography (TLC) on 0.25-mm pre-coated silica GF254 plates (Qingdong Yumingyuan silica gel reagent factory,

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Shandong, China, YUYUAN). Flash column chromatography was carried out with the indicated solvents using silica gel 60 (particle size 0.040-0.063 mm, Qingdong Yumingyuan silica gel reagent factory, Shandong, China, YUYUAN). Mass spectra were obtained on the API 4000 instrument (Applied Biosystems, Connecticut, USA) 1H nuclear magnetic resonance (1H NMR) spectra were

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recorded on the Bruker Avance DRX 400 spectrometer (Bruker, Switzerlands) using appropriate deuterated solvents and were expressed in parts per million (δ, ppm) downfield from

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tetramethylsilane (internal standard). Infrared (IR) spectra were recorded on the Nicolet Nexus 470FT-IR spectrometer (Wisconsin, USA) using KBr pellets. Melting points were uncorrected and determined with the X-6 melting point apparatus (Beijing Tianchengwode Biotech Co. Ltd, Beijing, China). Bacterial morphometric analysis was determined on the Olympus CKX41 microscope.

1

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IR, 1H-NMR and MS Spectra of Compound 3

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IR, 1H-NMR and MS Spectra of Compound 4

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IR, 1H-NMR and MS Spectra of Compound 5

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IR, 1H-NMR and MS Spectra of Compound 6

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IR, 1H-NMR and MS Spectra of Compound 7

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IR, 1H-NMR and MS Spectra of Compound 8

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IR, 1H-NMR and MS Spectra of Compound 9

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IR, 1H-NMR and MS Spectra of 10

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IR, 1H-NMR and MS Spectra of Compound 11

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IR, 1H-NMR and MS Spectra of Compound 12

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IR, 1H-NMR and MS Spectra of Compound 13

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IR, 1H-NMR and MS Spectra of Compound 14 13

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IR, 1H-NMR and MS Spectra of Compound 15

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IR, 1H-NMR and MS Spectra of Compound 16

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IR, 1H-NMR and MS Spectra of Compound 17

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IR, 1H-NMR and MS Spectra of Compound 18

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IR, 1H-NMR and MS Spectra of Compound 19 18

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H-NMR and MS Spectra of Compound 21

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H-NMR and MS Spectra of Compound 23

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H-NMR and MS Spectra of Compound 25

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IR, 1H-NMR and MS Spectra of Compound 26

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IR, 1H-NMR and MS Spectra of Compound 27

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IR, 1H-NMR and MS Spectra of Compound 28

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IR, 1H-NMR and MS Spectra of Compound 29

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H-NMR and MS Spectra of Compound 33

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IR, 1H-NMR and MS Spectra of Compound 34

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IR, 1H-NMR and MS Spectra of Compound 38

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IR, 1H-NMR and MS Spectra of Compound 39

38