European Journal of Medicinal Chemistry 90 (2015) 10e20

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

The synthesis and antibacterial activity of pyrazole-fused tricyclic diterpene derivatives Li-Gang Yu a, 1, Teng-Feng Ni b, 1, Wei Gao a, Yuan He c, Ying-Ying Wang a, Hai-Wei Cui a, Cai-Guang Yang b, Wen-Wei Qiu a, * a

Department of Chemistry, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China

b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 October 2014 Received in revised form 4 November 2014 Accepted 6 November 2014 Available online 6 November 2014

The diterpenoid compound 5 was identified as an antibacterial lead in our screening of small synthetic natural product-like (NPL) library. A series of novel diterpene derivatives were synthesized and investigated for their activity against Staphylococcus aureus Newman strain and multidrug-resistant strains (NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271). Among the compounds tested, 42 and 43 showed highest activity with a MIC of 1 mg/mL against strain Newman, 45 and 52 showed the most potent activity with MIC values of 0.71e3.12 mg/mL against five multidrug-resistant S. aureus. All highantimicrobial active compounds showed no obvious toxicity to human fibroblast (HAF) cells at the MIC concentration. © 2014 Published by Elsevier Masson SAS.

Keywords: Antibacterial Multidrug-resistant Diterpene derivative Cyclization reaction Synthesis

1. Introduction Infections caused by antibiotic-resistant Gram-positive bacteria are a serious public health problem around the world [1,2]. Methicillin-resistant Staphylococcus aureus (MRSA) encodes for a novel penicillin-binding protein PBP-2a and is resistant to b-lactam antibiotics, include penicillins, cephalosporins, monobactams and carbapenems [3,4]. Today, MRSA is the leading cause of bacterial infections resulting in increased mortality rates and hospitalization costs [5]. The incidence of infections caused by MRSA continues to increase [6,7], thus the demand for developing of new antimicrobial agents with novel structural classes and mechanisms is necessary. Diterpenoids are a large family of natural products exhibiting a wide range of biological activities, such as anti-inflammatory, antiHIV, anti-tumor, antidiabetic, and especially antibacterial activity [8,9]. For instance (Fig. 1), the jatrophane diterpenes

* Corresponding author. E-mail address: [email protected] (W.-W. Qiu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2014.11.015 0223-5234/© 2014 Published by Elsevier Masson SAS.

helioscopinolide A and B exhibit significant antibacterial activity against S. aureus [10], ferruginol, that has been isolated from the needles of the redwood Sequoia sempervirens strongly inhibits the growth of Gram-positive bacteria [11], and especially the wellknown natural diterpenoid totarol exhibits antibacterial activity against Gram-positive bacteria [12] and in particular MRSA [13e15]. Natural products still provide a rich resource for antibacterial drug discovery [16], however frequently the scarcity and poor activities of them limit their developments. Therefore the seeking of modified natural-based compounds and synthetic natural analogues are effective strategies in drug discovery. Herein, we report the discovery of an antibiotic lead in screening of one of our small synthetic natural product-like (NPL) library against S. aureus strain Newman. The novel small NPL library contains about 150 tricyclic diterpene analogues, which was constructed based on the cyclization reactions [17,18], and compound 5 (a pyrazole-fused tricyclic diterpene, Scheme 1) was chosen as a novel neuroprotective lead (MIC ¼ 4 mg/mL). A series of tricyclic diterpene analogues were synthesized based on this lead compound. Their in vitro antimicrobial activities against S. aureus strain Newman and five multidrug-resistant S. aureus were evaluated.

L.-G. Yu et al. / European Journal of Medicinal Chemistry 90 (2015) 10e20

Fig. 1. Chemical structures of antimicrobial diterpenoids.

2. Chemistry A series of pyrazole-fused tricyclic diterpene derivatives and their intermediates were synthesized according to the pathways described in Schemes 1e4. Lead compound 5 and its analogues (6e10) were synthesized as shown in Scheme 1. Coupling reaction of 6, 7-epoxygeranyl acetate with 4-methoxybenzylmagnesium chloride in the presence of Li2CuCl4 gave compound 1. The key intermediate 2 was obtained by cyclization of 1 under Lewis acid MeAlCl2 according to the similar procedure reported previously [17,18]. For the cyclization reaction, the StorkeEschenmoser hypothesis [19,20] shows that the preorganized chairechair conformation is important to the diastereoselectivity. The trans-trans-cyclized products with excellent region- and diastereoselectivity are produced by Lewis acidpromoted epoxide ring opening initiated the cascade antiparallel addition of olefins. According to the hypothesis, three chiral centers are formed in a single cyclization step, and only 2 out of 8 possible isomers are formed. The relative stereo-configuration of compound 2 was determined by X-ray analysis [21] (Fig. 2). Oxidation of 2 with 2-iodoxybenzoic acid (IBX) gave intermediate 3. Claisen condensation of 3 with ethyl formate produced compound 4, which was further reacted with hydrazine hydrate to produce compound 5. Derivatives 6e10 were synthesized by reaction of 5 with various acyl chlorides respectively. The intermediates (14, 23e27 and 31) for preparation of derivatives of 5 were synthesized as shown in Scheme 2. Treatment of compound 2 with acetyl chloride under DMAP gave compound 11. Compound 12 was afforded by Vilsmeier reaction of compound 11 with POCl3 and DMF. Refluxing of 12 with hydroxylamine hydrochloride and sodium formate in formic acid, then hydrolysis with NaOH in MeOH afforded intermediate 14. Compounds 19e22 were obtained by FriedeleCrafts acylations of 11 with various acyl

11

chlorides respectively, then hydrogenation with Pd/C under H2. Intermediates 23e26 were obtained by hydrolyzing of 19e22 with NaOH in a manner similar to that of 14. Bromination of compound 2 with Br2 in CH2Cl2 furnished intermediate 27. Compound 28 was produced by protection of the 3-hydroxyl group of 27 with TBSCl. Treatment of 28 with n-butyllithium, and then reaction with dry ice afforded compound 29. Intermediate 31 was prepared by deprotection of TBS group under boron trifluoride etherate and then esterification with EtOH in the presence of H2SO4. The intermediates (34, 38, and 39) for preparation of derivatives of 5 were synthesized as shown in Scheme 3. Compound 33 was obtained by Baeyer-Villiger oxidation of 15 with m-CPBA, and then hydrolysis with NaOH in MeOH. Intermediate 34 was prepared by benzyl etherification with BnBr in the presence of K2CO3. Hydrolysis of 15 with NaOH in MeOH afforded 35. Intermediate 38 was prepared by reduction of 35 with NaBH4, and then dehydration with TsOH in THF. Intermediate 39 was prepared by Grignard reaction of 35 with Methylmagnesium chloride, and then dehydration with TsOH in a manner similar to that of 38. The tricyclic diterpene derivatives (40e46 and 48e57) of 5 were synthesized as shown in Scheme 4. Compounds 40e49 were obtained in a manner similar to that of the lead compound 5, first by oxidation of the 3-hydroxyl group with IBX, then Claisen condensation with ethyl formate, finally condensation with hydrazine hydrate. Compound 50 was produced by hydrolyzing of 46 with NaOH in MeOH. Compounds 51 and 52 were obtained by hydrogenation of 47 and 49 with Pd/C under H2. Treatment 41, 45 and 52 with BBr3 respectively produced compounds 53e55. Compound 56 was obtained by acetylation of 53 with acetyl chloride. Etherification of 53 with allyl bromide in the presence of K2CO3 gave compound 57. 3. Biological evaluation The synthesized pyrazole-fused tricyclic diterpene derivatives 5e10, 40e46 and 48e57 were evaluated for their in vitro antibacterial activity (MIC; the minimum concentration of the compound that produced completely bacterial growth inhibition) against S. aureus strain Newman, and multidrug-resistant S. aureus NRS1(resistant to aminoglycosides and tetracycline), NRS-70 (resistant to erythromycin), NRS-100 (resistant to oxacillin and tetracycline), NRS-108 (resistant to gentamicin) and NRS-271 (linezolidresistant, containing phage type E-MRSA 15). They showed activity in the range of 0.71e30 mg/mL against the mentioned bacterial strains.

Scheme 1. Synthesis of lead compound 5 and its derivatives 6e10. Reagents and conditions: (a) 4-methoxybenzylmagnesium chloride, Li2CuCl4 (0.1 M in THF), THF, 0  C, 1 h, 69%; (b) MeAlCl2 (1 M in hexane), CH2Cl2, e78  C, 1 h, 68%; (c) IBX, DMSO, THF, rt, 3 h, 85%; (d) NaH, ethyl formate, toluene, rt, 2 h; (e) acetic acid, hydrazine hydrate, rt, 6 h, 83% (over two steps); (f) acyl chlorides, DMAP, CH2Cl2, rt, 6 h (54e88% for 6e10).

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Scheme 2. Synthesis of intermediates 14, 23e27 and 31. Reagents and conditions: (a) acetyl chloride, DMAP, CH2Cl2, rt, 4 h, 86%; (b) POCl3, DMF, CHCl3, reflux, 20 h, 60%; (c) hydroxylamine hydrochloride, HCOONa, HCOOH, reflux, 8 h, 73% for 13; (d) acyl chlorides, AlCl3, CH2Cl2, 0  C, 2 h (81e99% for 15e18); (e) Pd/C, H2, acetic acid, 60  C, 6 h (for 19e22); (f) NaOH, MeOH, reflux, 3 h, 89% for 14 and 39e73% (over two steps) for 23e26; (g) Br2, CH2Cl2, 0  C, 1 h, 96%; (h) TBSCl, imidazole, DMF, rt, 12 h, 95%; (i) n-BuLi, CO2, THF, 78  C, 2 h, 77%; (j) boron trifluoride etherate, CHCl3, rt, 1 h, 79%; (k) EtOH, H2SO4, reflux, 2 h, 98%.

Scheme 3. Synthesis of intermediates 34, 38, and 39. Reagents and conditions: (a) mCPBA, CH2Cl2, rt, 12 h; (b) MeOH, NaOH, reflux, 3 h, 72% for 33 (over two steps) and 99% for 35; (c) BnBr, K2CO3, DMF, rt, 12 h, 77%; (d) NaBH4, MeOH, rt, 3 h, for 36; MeMgCl (3 M in THF), THF, rt, 6 h, for 37; (e) p-TsOH, THF, reflux, 5 h, 75% for 38 and 71% for 39 (over two steps).

4. Result and discussion 4.1. Antibacterial activity The first round synthetic analogues were obtained by modification of the pyrazole ring of lead compound 5 with various acyl chlorides, and evaluated for antibacterial activity by testing minimum inhibitory concentrations (MICs, Table 1) against S. aureus strain Newman. All analogues showed decreased antibacterial activity compared to 5, and most of them showed no inhibition of growth, except compound 9 (modified with nicotinoyl group). These results demonstrate that the pyrazole ring of compound 5 is not suitable for modification. The second round synthetic analogues were obtained by modification of the aromatic ring of lead compound 5 and evaluated against S. aureus strain Newman and multidrug-resistant S. aureus NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271 (Table 2). Totarol was used as a positive control.

For S. aureus strain Newman, the MICs were between 1 and 30 mg/mL. The benzene ring with hydrophobic substituents that is ethyl (41), propyl (42), butyl (43), bromide (45), vinyl (48), isopropenyl (49) and isopropyl (52) exhibited more potent activity than 5. Due to the large n-octyl group, compound 44 showed completely inactive against all testing bacterial strains. Compounds 42 and 43 showed highest activity (1 mg/mL), which was four-fold more active than 5, and slightly more potent than totarol. Analogues with hydrophilic substituents, that is, cyano (40), hydroxyl (51), carboxyl (50) and its corresponding ethyl formate (46) showed no inhibition (>30 mg/mL) at all. Analogues with the methoxy group hydrolyzed to hydroxyl group (53e55) or transformed into ethyl acetate (56) and allyl ether (57) decreased the inhibitory effects obviously compared to corresponding compounds (41, 45 and 52) with methoxy group. For S. aureus NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271, the MICs were between 0.71 and 30 mg/mL. Compounds 45 and 52 showed the most potent activity which was about 5e21 folds more potent than 5, and 1e2 folds more potent than totarol. In general, to these multidrug-resistant S. aureus, small hydrophobic substituents such as ethyl, bromide, vinyl, isopropenyl and isopropyl were more favorable than the relatively large substituents such as propyl, butyl and n-octyl (41, 45, 48, 49 and 52 vs 42e44), except compound 48 for NRS-100. Analogues with the methoxy group hydrolyzed to hydroxyl group (53e55) or transformed into ethyl acetate (56) and allyl ether (57) showed decreased the antimicrobial activity dramatically (>30 mg/mL) compared to 41, 45 and 52, which was similar for S. aureus strain Newman. The SARs of these compounds revealed that the antibacterial activity was enhanced by the introduction of small hydrophobic substituents into the aromatic ring of lead compound 5. Previous study demonstrated that the hydroxyl of totarol and its synthetic analogues was important and should be kept, and if it was converted into methoxyl, the antimicrobial activity would be completely removed [22,23]. On the contrary, the methoxyl was a key substituent group for possessing high activity of our synthetic tricyclic diterpene derivatives and if it was hydrolyzed into hydroxyl, the antibacterial activity will be decreased dramatically. The results revealed that these synthetic diterpenoids perhaps shared different

L.-G. Yu et al. / European Journal of Medicinal Chemistry 90 (2015) 10e20

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Scheme 4. Synthesis of derivatives 40e46 and 48e57. Reagents and conditions: (a) IBX, DMSO, THF, rt, 3 h; (b) NaH, ethyl formate, toluene, rt, 2 h; (c) acetic acid, hydrazine hydrate, rt, 6 h, 43e86% for 40e49 (over three steps); (d) NaOH, THF, reflux, 3 h, 90%; (e) Pd/C, MeOH, rt, 12 h, 89% for 51 and 83% for 52. (f) BBr3, CH2Cl2, 78  C, 57e84% for 53e55; (g) 1). AcCl, DMAP, CH2Cl2, rt, 6 h; 2). MeOH, TEA, 2 h, 77%; (h) allyl bromide, K2CO3, DMF, rt, 10 h, 56%.

Fig. 2. Single-crystal X-ray structure of cyclization product 2 (thermal ellipsoids at 50% probability).

Table 1 Preliminary MICs of compound 5 and its pyrazole ring-modified analogues against S. aureus strain Newman. Compound

MIC(mg/mL)

Compound

MIC(mg/mL)

5 6 7

4 >30 >30

8 9 10

>30 6 >30

2.4e4.2 times lower than totarol. Besides, in order to further examine the toxicity, compounds 5, 45, 52 and totarol were selected to investigate the cellular morphology of HAF under the MIC (1.56 mg/mL) and the average IC50 (30 mg/mL) concentrations. After incubation for 24 h, the compounds treated at 1.56 mg/mL dose did not exhibit any toxicity to HAF cells and showed normal morphology (Fig. 3). However, when the cells were exposed to 30 mg/mL concentration of the tested compounds, they lost normal morphology (Fig. 3). The above cytotoxicity assay demonstrated that our compounds bearing good selectivity for bacterial over mammalian cells.

5. Conclusion For the first time, a small synthetic diterpenoids NPL library was constructed and a promising antibacterial lead compound 5 was Table 2 MICs of synthetic tricyclic diterpene derivatives against S. aureus strain Newman (Newman), S. aureus NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271. Compound

mechanism with totarols for antibacterial activity, and details should be further explored.

4.2. In vitro toxicity study One of the major hindrances for druggability of many compounds with effective antibacterial activities is their toxicity to normal cells. Thus, it is important to measure cytotoxicity in novel antibiotics discovery. The lead (5) and compounds (41, 42, 43, 45, 48, 49 and 52) with low MIC against Newman or NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271 were tested their cytotoxic activity on a human fibroblast (HAF) cell line using the MTS assay to prove the potency of the compounds. The cytotoxicity results of these compounds on normal mammalian cells were summarized in Table 3. It can be seen from the table that these tested compounds (except 43), showed low cytotoxicity with IC50 20e36 mg/mL, which was

5 40 41 42 43 44 45 46 48 49 50 51 52 53 54 55 56 57 Totarol

MIC(mg/mL) Newman

NRS-1

NRS-70

NRS-100

NRS-108

NRS-271

4 >30 1.25 1 1 >30 3.12 >30 3.12 1.56 >30 >30 1.56 6.25 6.25 6.25 6.25 3.12 1.56

15 e 7.5 >30 >30 >30 1.56 e 12.5 6.25 e >30 1.56 >30 >30 >30 >30 >30 3.12

15 e 8 24 >30 >30 1.56 e 6.25 6.25 e >30 0.71 >30 >30 >30 >30 >30 1.56

12 e 4 3.75 6 >30 1.56 e 6.25 3.13 e >30 1.56 >30 >30 >30 >30 >30 1.56

15 e 7.5 >30 >30 >30 1.56 e 6.25 6.25 e >30 1.56 >30 >30 >30 >30 >30 3.12

15 e 8 >30 >30 >30 1.56 e 6.25 12.5 e >30 3.12 >30 >30 >30 >30 >30 3.12

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L.-G. Yu et al. / European Journal of Medicinal Chemistry 90 (2015) 10e20

Table 3 IC50 values of synthetic tricyclic diterpene derivatives against the growth of HAF. Compound

IC50(mg/mL)

5 41 42 43 45

34.65 30.13 20.08 9.37 33.65

± ± ± ± ±

1.56 2.45 1.85 1.54 2.87

Compound

IC50(mg/mL)

48 49 52 Totarol

22.20 35.95 23.27 8.46

± ± ± ±

1.95 2.82 1.02 0.87

screened out from this novel library. A series of new analogues were synthesized based on the lead compound and investigated for their antibacterial activity in vitro against S. aureus strain Newman and five multidrug-resistant S. aureus (NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271). For these Gram-positive bacteria species, the synthetic derivatives showed antibacterial activity with MIC values of 0.71e30 mg/mL. For Newman, compounds 42 and 43 showed highest activity (MIC, 1 mg/mL) and for multidrug-resistant bacteria, compounds 45 and 52 exhibited the most potent activity (MIC, 0.71e3.12 mg/mL). All high active antimicrobial compounds did not show any obvious toxicity to human adult fibroblast (HAF) cells at MIC dose (Table 3 and Fig. 3). Here, we offered a series of promising novel tricyclic diterpene derivatives for discovery of new chemical entities against multidrug-resistant bacteria. Further indepth SAR study of the active scaffolds and the mechanism research are also required. 6. Experimental section 6.1. General All reagents and chemicals were purchased from commercial suppliers and used without further purification unless otherwise stated. When needed, the reactions were carried out in flame or oven-dried glassware under a positive pressure of dry N2. Column chromatography was performed on silica gel (QinDao, 200e300 mesh) using the indicated eluents. Thin-layer chromatography was carried out on silica gel plates (QinDao) with a layer thickness of 0.25 mm. Melting points were determined using the MEL-TEMP 3.0 apparatus and uncorrected. 1H (300, 400 and 500 MHz) and 13C (100 and 125 MHz) NMR spectra were recorded on Varian Mercury300, Bruker AM-400 and Bruker AV-500 spectrometer with CDCl3 or DMSO-d6 as solvent and tetramethylsilane (TMS) as the internal standard. All chemical shift values were reported in units of d (ppm). The following abbreviations were used to indicate the

peak multiplicity: s ¼ singlet; d ¼ doublet; t ¼ triplet; m ¼ multiplet; br ¼ broad. High-resolution mass data were obtained on a Bruker microOTOF-Q II spectrometer. X-ray crystallography analysis was performed on a Bruker SMART ApexII X-ray diffractionmeter.

6.2. Synthesis of lead compound 5 To a solution of 6,7-epoxygeranyl acetate (9.04 g, 42.6 mmol) in THF (60 mL) at 0  C was added Li2CuCl4 (34 mL, 0.1 M solution in THF). Then a solution of 4-Methoxybenzylmagnesium chloride (256 mL, 0.25 M solution in THF) was added in drops over 30 min and stirring was continued for 1 h at 0  C. The reaction mixture was quenched with half saturated aqueous NH4Cl (200 mL), extracted with AcOEt (3  50 mL). The combined organic extract was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 20/1 v/v) to give compound 1 (8.1 g, 69%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d 7.10 (d, J ¼ 8.3 Hz, 2 H), 6.82 (d, J ¼ 8.3 Hz, 2 H), 5.22 (t, J ¼ 6.8 Hz, 1 H), 3.79 (s, 3 H), 2.69 (t, J ¼ 6.2 Hz, 1 H), 2.59 (t, J ¼ 7.7 Hz, 2 H), 2.35e2.02 (m, 4 H), 1.67e1.59 (m, 2 H), 1.58 (s, 3 H), 1.28 (d, J ¼ 15.6 Hz, 6 H). To a solution of compound 1 (4.1 g, 15 mmol) in dry CH2Cl2 (200 mL) at 78  C was added MeAlCl2 (22.5 mL, 1 M in hexane) in drops over 1.5 h under N2. After the addition was complete, the mixture was stirred for another 1 h, Et3N (5 mL) was added, followed by MeOHeH2O solvent (10 mL, 4:1) at 78  C. The resulting mixture was warmed to room temperature and then added dropwise saturated NH4Cl (120 mL). After separation of the layers, the aqueous phase was extracted with CH2Cl2 (3  30 mL). The combined organic extract was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 5/1 v/v) to give compound 2 (2.8 g, 68%) as a white solid. 1H NMR (500 MHz, CDCl3) d 6.97 (d, J ¼ 8.4 Hz, 1 H), 6.79 (d, J ¼ 2.6 Hz, 1 H), 6.67 (dd, J ¼ 8.4, 2.7 Hz, 1 H), 3.77 (s, 3 H), 3.31 (dd, J ¼ 11.4, 4.8 Hz, 1 H), 3.00e2.67 (m, 2 H), 2.28 (dt, J ¼ 13.1, 3.5 Hz, 1 H), 1.93e1.67 (m, 4 H), 1.56 (td, J ¼ 13.1, 4.1 Hz, 1 H), 1.45 (s, 1 H), 1.32 (dd, J ¼ 12.3, 2.3 Hz, 1 H), 1.23e1.16 (m, 3 H), 1.07 (s, 3 H), 0.90 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 157.78, 150.69, 129.81, 127.30, 111.05, 110.27, 78.70, 55.31, 49.83, 39.08, 37.86, 37.02, 29.88, 28.25, 28.04, 24.85, 19.00, 15.49. To a solution of compound 2 (548 mg, 2.0 mmol) in THF-DMSO mixed solvent (10 mL, 1:2) was added IBX (1.1 g, 4.0 mmol). The reaction mixture was stirred for 3 h at room temperature, then was

Fig. 3. Normal growth (control) of human fibroblast (HAF) cells and morphological changes in the cells at concentrations of 1.56 mg/mL and 30 mg/mL compounds 5, 45, 52 and totarol.

L.-G. Yu et al. / European Journal of Medicinal Chemistry 90 (2015) 10e20

added with H2O (20 mL) and AcOEt (20 mL). After separation of the layers, the aqueous phase was extracted with AcOEt (2  10 mL). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 10/1 v/v) to give compound 3 (462 mg, 85%) as a white solid. To a solution of compound 3 (250 mg，0.92 mmol) in toluene (20 mL) was added NaH (183 mg, 60%, 4.6 mmol). After stirring for 0.5 h at room temperature, ethyl formate (681 mg, 9.2 mmol) was added and the reaction mixture was stirred for another 2 h. Toluene was removed and ice water (10 mL) was added, then the aqueous phase was extracted with AcOEt (3  10 mL). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated to give compound 4 as a yellow oil and used for next step without further purification. 1H NMR (300 MHz, CDCl3) d 14.99 (d, J ¼ 3.1 Hz, 1 H), 8.79 (d, J ¼ 2.8 Hz, 1 H), 7.02 (d, J ¼ 8.6 Hz, 1 H), 6.87 (d, J ¼ 2.5 Hz, 1 H), 6.73 (dd, J ¼ 8.4, 2.6 Hz, 1 H), 3.81 (s, 3 H), 2.97e2.69 (m, 3 H), 2.42 (d, J ¼ 14.4 Hz, 1 H), 1.89e1.65 (m, 3 H), 1.28 (s, 3 H), 1.21 (d, J ¼ 4.3 Hz, 6 H). To a solution of compound 4 in acetic acid (5 mL) was added 85% hydrazinium hydrate solution (280 mg, 5.6 mmol). The reaction mixture was stirred for 6 h at room temperature and then AcOEt (30 mL) was added. The mixture was washed with saturated NaHCO3, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (DCM/MeOH, 50/1 v/v) to give compound 5 (226 mg, 83% over two steps) as a yellow solid; mp: 187e188  C. 1H NMR (400 MHz, CDCl3) d 7.42 (s, 1 H), 7.03 (d, J ¼ 8.4 Hz, 1 H), 6.91 (d, J ¼ 2.5 Hz, 1 H), 6.73 (dd, J ¼ 8.4, 2.6 Hz, 1 H), 3.82 (s, 3 H), 3.15 (d, J ¼ 14.9 Hz, 1 H), 3.00e2.69 (m, 2 H), 2.55 (d, J ¼ 14.8 Hz, 1 H), 1.95 (dd, J ¼ 12.6, 5.6 Hz, 1 H), 1.87e1.66 (m, 2 H), 1.43 (s, 3 H), 1.34 (s, 3 H), 1.20 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 158.12, 150.40, 148.37, 133.34, 129.89, 127.93, 113.14, 111.65, 111.62, 55.43, 50.11, 39.60, 35.65, 33.94, 31.57, 30.83, 24.83, 23.64, 20.10. HRMS(ESI): calcd for C19H25N2O[M þ H]þ; 297.1967; found 297.1993. 6.3. General procedure for the synthesis of compounds 6e10 To a solution of 5 (89 mg, 0.3 mmol) and DMAP (122 mg, 1 mmol) in dry DCM (10 mL) was added acyl chloride (1 mmol) under N2. The reaction mixture was stirred for 6 h at room temperature and then concentrated. The residue was purified by column chromatography (petroleum ether/AcOEt, 5/1 v/v) to afford the desired product. 6.3.1. Compound 6 White solid; yield: 82%; mp: 128e129  C. 1H NMR (400 MHz, CDCl3) d 7.98 (s, 1 H), 7.02 (d, J ¼ 8.4 Hz, 1 H), 6.72 (dd, J ¼ 8.4, 2.5 Hz, 1 H), 3.81 (s, 3 H), 3.18 (d, J ¼ 15.3 Hz, 1 H), 2.98e2.72 (m, 2 H), 2.67 (s, 3 H), 2.51 (d, J ¼ 15.3 Hz, 1 H), 1.95 (dd, J ¼ 12.5, 5.7 Hz, 1 H), 1.84e1.66 (m, 2 H), 1.40 (s, 3 H), 1.33 (s, 3 H), 1.18 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 169.73, 162.63, 158.17, 147.68, 130.00, 128.01, 125.08, 119.41, 111.79, 111.63, 55.45, 50.17, 39.20, 35.81, 35.04, 31.67, 30.82, 24.73, 24.17, 21.74, 20.31; HRMS(ESI): calcd for C21H26N2NaO2[M þ Na]þ; 361.1892; found 361.1914. 6.3.2. Compound 7 White solid; yield: 88%; mp: 122e124  C. 1H NMR (400 MHz, CDCl3) d 7.98 (s, 1 H), 7.02 (d, J ¼ 8.4 Hz, 1 H), 6.89 (d, J ¼ 2.0 Hz, 1 H), 6.72 (dd, J ¼ 8.3, 2.0 Hz, 1 H), 3.81 (s, 3 H), 3.22e3.00 (m, 3 H), 3.00e2.72 (m, 2 H), 2.51 (d, J ¼ 15.1 Hz, 1 H), 1.95 (dd, J ¼ 12.4, 5.4 Hz, 1 H), 1.86e1.66 (m, 2 H), 1.40 (s, 3 H), 1.33 (s, 3 H), 1.27 (d, J ¼ 8.6 Hz, 3 H), 1.18 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 173.18, 162.38, 158.17, 147.74, 130.01, 128.03, 125.16, 119.01, 111.80, 111.64, 55.47, 50.19, 39.22, 35.83, 35.04, 31.69, 30.84, 27.48, 24.75, 24.19,

15

20.32, 8.72; HRMS(ESI): calcd for C22H28N2NaO2 [M þ Na]þ; 375.2048; found 375.2041. 6.3.3. Compound 8 White solid; yield: 54%; mp: 149e150  C. 1H NMR (400 MHz, CDCl3) d 7.76 (s, 1 H), 7.03 (d, J ¼ 8.4 Hz, 1 H), 6.87 (d, J ¼ 2.5 Hz, 1 H), 6.73 (dd, J ¼ 8.4, 2.5 Hz, 1 H), 3.81 (s, 3 H), 3.28 (s, 3 H), 3.18 (d, J ¼ 15.2 Hz, 1 H), 3.01e2.70 (m, 2 H), 2.51 (d, J ¼ 15.2 Hz, 1 H), 1.95 (dd, J ¼ 12.5, 5.6 Hz, 1 H), 1.88e1.65 (m, 2 H), 1.42 (s, 3 H), 1.35 (s, 3 H), 1.18 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 164.10, 158.16, 147.45, 130.04, 128.06, 127.98, 118.15, 111.73, 111.66, 55.44, 50.15, 41.19, 39.11, 35.55, 35.22, 31.62, 30.76, 24.70, 24.11, 20.25; HRMS(ESI): calcd for C20H27N2OS[M þ H]þ; 375.1742; found 375.1756. 6.3.4. Compound 9 White solid; yield: 87%; mp: 142e144  C. 1H NMR (400 MHz, CDCl3) d 9.50 (s, 1 H), 8.82 (s, 1 H), 8.62 (d, J ¼ 7.8 Hz, 1 H), 8.18 (s, 1 H), 7.58e7.50 (m, 1 H), 7.03 (d, J ¼ 8.4 Hz, 1 H), 6.90 (d, J ¼ 2.3 Hz, 1 H), 6.74 (dd, J ¼ 8.3, 2.4 Hz, 1 H), 3.82 (s, 3 H), 3.27 (d, J ¼ 15.5 Hz, 1 H), 2.97e2.76 (m, 2 H), 2.57 (d, J ¼ 15.5 Hz, 1 H), 1.96 (dd, J ¼ 12.6, 5.5 Hz, 1 H), 1.85 (d, J ¼ 12.8 Hz, 1 H), 1.78e1.69 (m, 1 H), 1.42 (s, 3 H), 1.34 (s, 3 H), 1.21 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 164.34, 164.06, 158.18, 152.79, 152.76, 147.49, 139.43, 130.04, 128.41, 127.98, 127.05, 122.96, 119.86, 111.80, 111.66, 55.46, 50.09, 39.22, 35.77, 35.14, 31.68, 30.78, 29.82, 24.81, 24.24, 20.29; HRMS(ESI): calcd for C25H28N3O2 [M þ H]þ; 402.2182; found 402.2203. 6.3.5. Compound 10 White solid; yield: 79%; mp 128e131  C. 1H NMR (400 MHz, CDCl3) d 8.84 (d, J ¼ 4.9 Hz, 2 H), 8.16 (s, 1 H), 8.11 (d, J ¼ 5.5 Hz, 2 H), 7.04 (d, J ¼ 8.4 Hz, 1 H), 6.90 (d, J ¼ 2.4 Hz, 1 H), 6.74 (dd, J ¼ 8.4, 2.4 Hz, 1 H), 3.82 (s, 3 H), 3.27 (d, J ¼ 15.5 Hz, 1 H), 2.97e2.76 (m, 2 H), 2.57 (d, J ¼ 15.6 Hz, 1 H), 1.96 (dd, J ¼ 12.8, 5.6 Hz, 1 H), 1.87e1.70 (m, 2 H), 1.41 (s, 3 H), 1.33 (s, 3 H), 1.21 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 164.50, 164.36, 158.22, 150.02, 147.44, 139.55, 130.09, 128.01, 127.07, 125.01, 120.28, 111.82, 111.71, 55.50, 50.11, 39.23, 35.80, 35.18, 31.70, 30.79, 24.83, 24.28, 20.31. HRMS(ESI): calcd for C25H28N3O2[M þ H]þ; 402.2182; found 402.2192. 6.4. Synthesis of compound 14 To a solution of compound 2 (2.65 g, 10.9 mmol) and DMAP (244 mg, 2 mmol) in DCM (30 mL) was added Ac2O (3.3 g, 32.6 mmol). The reaction mixture was stirred for 4 h at room temperature, and then concentrated. The residue was purified by column chromatography (petroleum ether/AcOEt, 20/1 v/v) to give compound 11 (2.68 g, 86%) as a white solid. 1H NMR (400 MHz, CDCl3) d 6.97 (d, J ¼ 8.4 Hz, 1 H), 6.77 (d, J ¼ 2.6 Hz, 1 H), 6.67 (dd, J ¼ 8.4, 2.6 Hz, 1 H), 4.56 (dd, J ¼ 11.5, 4.8 Hz, 1 H), 3.77 (s, 3 H), 2.96e2.73 (m, 2 H), 2.35e2.20 (m, 1 H), 2.07 (s, 3 H), 1.93e1.61 (m, 5 H), 1.40 (dd, J ¼ 12.2, 2.3 Hz, 1 H), 1.22 (s, 3 H), 0.96 (d, J ¼ 5.9 Hz, 6 H). DMF (9.24 g, 126 mmol) was added to POCl3 (20 mL) at 0  C. After stirring for 1 h, a solution of compound 11 (1.0 g, 3.2 mmol) in CHCl3 (10 mL) was added in drops and then the reaction mixture was heated to reflux for 20 h. After cooling to room temperature, the mixture was poured into ice water (100 mL) and extracted with EtOAc (30 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 5/1 v/v) to give compound 12 (656 mg, 60%) as a white solid. 1H NMR (400 MHz, CDCl3) d 10.37 (s, 1 H), 7.51 (s, 1 H), 6.81 (s, 1 H), 4.55 (dd, J ¼ 11.5, 4.6 Hz, 1 H), 3.88 (s, 3 H), 3.02e2.71 (m, 2 H), 2.32 (s, 1 H), 2.08 (s, 3 H), 1.96e1.63 (m, 5 H), 1.39 (dd, J ¼ 12.2, 2.1 Hz, 1 H), 1.24 (s, 3 H), 0.97 (d, J ¼ 6.3 Hz, 6 H).

16

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A solution of compound 12 (200 mg, 0.58 mmol), HCOONa (592 mg, 8.7 mmol) and NH2OH$HCl (202 mg, 2.9 mmol) in HCOOH (10 mL) was heated to reflux for 8 h under N2. After cooling to room temperature, the mixture was poured into water (30 mL) and extracted with EtOAc (20 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 2/1 v/v) to give compound 13 (145 mg, 73%) as a yellow solid. To a solution of compound 13 (145 mg, 0.42 mmol) in MeOH (10 mL) was added NaOH (80 mg, 2.0 mmol). The reaction mixture was heated to reflux for 3 h and then concentrated. Water (10 mL) was added to the residue and extracted with EtOAc (10 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 2/1 v/v) to give compound 14 (112 mg, 89%) as a yellow solid. 1H NMR (500 MHz, CDCl3) d 7.23 (s, 1H), 6.80 (s, 1 H), 3.88 (s, 3 H), 3.31 (dd, J ¼ 11.5, 4.5 Hz, 1 H), 2.95e2.70 (m, 2 H), 2.27 (dt, J ¼ 12.9, 3.2 Hz, 1 H), 1.96e1.66 (m, 5 H), 1.28 (d, J ¼ 12.3 Hz, 1 H), 1.20 (s, 3 H), 1.08 (s, 3 H), 0.90 (s, 3 H). 6.5. General procedure for the synthesis of compounds 15e18 To a solution of 11 (722 mg, 2.28 mmol) in DCM (30 mL) was added AlCl3 (913 mg, 6.84 mmol) and acyl chloride (4.56 mmol) at 0  C. The reaction mixture was stirred for 2 h at 0  C and then water (20 mL) was added. After the separation of organic layer, the aqueous phase was extracted with DCM (2  15 mL). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 10/1 v/v) to afford the desired products. 6.5.1. Compound 15 White solid; yield: 81%. 1H NMR (300 MHz, CDCl3) d 7.45 (s, 1 H), 6.79 (s, 1 H), 4.55 (dd, J ¼ 11.2, 4.8 Hz, 1 H), 3.87 (s, 3 H), 3.05e2.64 (m, 2 H), 2.58 (s, 3 H), 2.29 (d, J ¼ 12.9 Hz, 1 H), 2.08 (s, 3 H), 1.97e1.61 (m, 5 H), 1.23 (s, 4 H), 0.97 (d, J ¼ 4.3 Hz, 6 H). 6.5.2. Compound 16 White solid; yield: 85%. 1H NMR (300 MHz, CDCl3) d 7.39 (s, 1 H), 6.78 (s, 1 H), 4.55 (dd, J ¼ 11.2, 4.7 Hz, 1 H), 3.85 (s, 3 H), 3.04e2.85 (m, 3 H), 2.86e2.69 (m, 1 H), 2.29 (d, J ¼ 12.8 Hz, 1 H), 2.08 (s, 3 H), 1.87 (t, J ¼ 12.3 Hz, 3 H), 1.80e1.62 (m, 3 H), 1.38 (d, J ¼ 12.0 Hz, 1 H), 1.23 (s, 3 H), 1.14 (t, J ¼ 7.3 Hz, 3 H), 0.97 (d, J ¼ 4.6 Hz, 6 H). 6.5.3. Compound 17 White solid; yield: 91%. 1H NMR (400 MHz, CDCl3) d 7.36 (s, 1 H), 6.77 (s, 1 H), 4.55 (dd, J ¼ 11.5, 4.6 Hz, 1 H), 3.85 (s, 3 H), 3.05e2.67 (m, 4 H), 2.29 (d, J ¼ 12.9 Hz, 1 H), 2.08 (s, 3 H), 1.96e1.62 (m, 7 H), 1.38 (dd, J ¼ 12.1, 1.5 Hz, 1 H), 1.22 (s, 3 H), 1.03e0.90 (m, 9 H). 6.5.4. Compound 18 White solid; yield: 99%. 1H NMR (400 MHz, CDCl3) d 7.36 (s, 1 H), 6.77 (s, 1 H), 4.55 (dd, J ¼ 11.5, 4.6 Hz, 1 H), 3.84 (s, 3 H), 3.01e2.69 (m, 4 H), 2.38e2.20 (m, 1 H), 2.08 (s, 3 H), 1.95e1.70 (m, 4 H), 1.42e1.24 (m, 9 H), 1.22 (s, 3 H), 0.96 (d, J ¼ 6.2 Hz, 6 H), 0.87 (t, J ¼ 6.7 Hz, 3 H). 6.6. General procedure for the synthesis of compounds 23e26 One of compounds 15e18 (1.15 mmol) was dissolved in CH3COOH (8 mL), 5% Pd on carbon (40 mg) was added. The reaction mixture was subjected to 1 atm of H2 and stirred for 6 h at 60  C.

After cooling, AcOEt (30 mL) was added and the reaction mixture was filtered. The filtrate was washed by saturated NaHCO3 (3  20 mL) and concentrated to give the crude compounds 19e22. One of compounds 19e22 was dissolved in MeOH (10 mL), NaOH (80 mg, 2.0 mmol) was added in. The reaction mixture was heated to reflux for 3 h and then concentrated. Water (10 mL) was added to the residue and extracted with EtOAc (10 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 5/1 v/v) to afford the desired products. 6.6.1. Compound 23 White solid; yield: 64% (over two steps). 1H NMR (400 MHz, CDCl3) d 6.82 (s, 1 H), 6.70 (s, 1 H), 3.79 (s, 3 H), 3.31 (dd, J ¼ 11.0, 5.0 Hz, 1 H), 2.95e2.70 (m, 2 H), 2.66e2.47 (m, 2 H), 2.29 (dt, J ¼ 12.9, 3.2 Hz, 1 H), 1.93e1.65 (m, 4 H), 1.63e1.55 (m, 1 H), 1.33 (dd, J ¼ 12.1, 1.6 Hz, 1 H), 1.21 (s, 3 H), 1.17 (t, J ¼ 7.5 Hz, 3 H), 1.07 (s, 3 H), 0.90 (s, 3 H). 6.6.2. Compound 24 Colorless oil; yield: 42% (over two steps). 1H NMR (400 MHz, CDCl3) d 6.80 (s, 1 H), 6.70 (s, 1 H), 3.78 (s, 3 H), 3.31 (dd, J ¼ 11.0, 5.1 Hz, 1 H), 2.98e2.67 (m, 2 H), 2.63e2.38 (m, 2 H), 2.29 (dt, J ¼ 12.9, 3.3 Hz, 1 H), 1.95e1.65 (m, 5 H), 1.62e1.54 (m, 3 H), 1.33 (dd, J ¼ 12.2, 2.0 Hz, 1 H), 1.21 (s, 3 H), 1.07 (s, 3 H), 0.95 (t, J ¼ 7.3 Hz, 3 H), 0.90 (s, 3 H). 6.6.3. Compound 25 Colorless oil; yield: 73% (over two steps). 1H NMR (400 MHz, CDCl3) d 6.79 (s, 1 H), 6.69 (s, 1 H), 3.78 (s, 3 H), 3.31 (d, J ¼ 6.7 Hz, 1 H), 2.93e2.64 (m, 2 H), 2.64e2.41 (m, 2 H), 2.29 (dt, J ¼ 12.9, 3.2 Hz, 1 H), 1.93e1.66 (m, 4 H), 1.41e1.29 (m, 4 H), 1.21 (s, 3 H), 1.07 (s, 3 H), 0.99e0.75 (m, 6 H). 6.6.4. Compound 26 Colorless oil; yield: 39% (over two steps). 1H NMR (400 MHz, CDCl3) d 6.79 (s, 1 H), 6.69 (s, 1 H), 3.78 (s, 3 H), 3.31 (dd, J ¼ 11.0, 5.0 Hz, 1 H), 3.00e2.67 (m, 2 H), 2.63e2.40 (m, 2 H), 2.28 (d, J ¼ 13.0 Hz, 1 H), 1.92e1.64 (m, 4 H), 1.39e1.22 (m, 12 H), 1.21 (s, 3 H), 1.07 (s, 3 H), 0.93e0.80 (m, 6 H). 6.7. Synthesis of compounds 27 To a solution of compound 2 (831 mg, 3.0 mmol) in DCM (80 mL) was added bromine (485 mg, 3.0 mmol) in batches at 0  C. The reaction mixture was stirred for 1 h at 0  C and then poured into saturated NaHSO3. After the separation of organic layer, the aqueous phase was extracted with DCM (2  15 mL). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated to give compound 27 (1.03 g, 96%) as a white solid. 1H NMR (300 MHz, CDCl3) d 7.21 (s, 1 H), 6.75 (s, 1 H), 3.85 (s, 3 H), 3.31 (dd, J ¼ 11.0, 5.0 Hz, 1 H), 2.95e2.71 (m, 2 H), 2.27 (d, J ¼ 13.0 Hz, 1 H), 1.92e1.55 (m, 7 H), 1.19 (s, 3 H), 1.07 (s, 3 H), 0.89 (s, 3 H). 6.8. Synthesis of compounds 31 To a solution of compound 27 (10.69 g, 30.33 mmol) in DMF (60 mL) was added TBSCl (6.86 g, 45.5 mmol) and imidazole (4.13 g, 60.66 mmol). The reaction mixture was stirred for 12 h at room temperature and then poured into water (100 mL), extracted with AcOEt (40 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 20/1 v/v) to give compound 28 (13.38 g, 95%) as a white solid. 1H NMR

L.-G. Yu et al. / European Journal of Medicinal Chemistry 90 (2015) 10e20

(400 MHz, CDCl3) d 7.20 (s, 1 H), 6.76 (s, 1 H), 3.84 (s, 3 H), 3.26 (dd, J ¼ 11.3, 4.6 Hz, 1 H), 2.96e2.64 (m, 2 H), 2.21 (dt, J ¼ 12.9, 3.4 Hz, 1 H), 1.93e1.62 (m, 4 H), 1.18 (s, 3 H), 0.98 (s, 3 H), 0.91 (s, 9 H), 0.85 (s, 3 H), 0.05 (d, J ¼ 7.0 Hz, 6 H). To a solution of compound 28 (7.0 g, 15 mmol) in dry THF (100 mL) under N2 was added n-BuLi (8.25 mL, 2 M in hexane) in drops at 78  C. After the addition was complete, the mixture was stirred for another 0.5 h. Then excessive dry CO2 was bubbled in at 78  C for 2 h. When rising to room temperature, AcOEt (50 mL) and hydrochloric acid (50 mL, 0.5 M) was added. After the separation of organic layer, the aqueous phase was extracted with AcOEt (40 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 2/1 v/v) to give compound 29 (5.0 g, 77%) as a white solid. 1H NMR (400 MHz, CDCl3) d 10.71 (s, 1 H), 7.85 (s, 1 H), 6.88 (s, 1 H), 4.02 (s, 3 H), 3.26 (dd, J ¼ 11.3, 4.5 Hz, 1 H), 3.04e2.63 (m, 2 H), 2.22 (d, J ¼ 12.9 Hz, 1 H), 1.96e1.67 (m, 4 H), 1.30e1.24 (m, 1 H), 1.20 (s, 3 H), 0.99 (s, 3 H), 0.91 (s, 9 H), 0.87 (s, 3 H), 0.05 (d, J ¼ 6.6 Hz, 6 H); 13C NMR (100 MHz, CDCl3) d 165.83, 157.47, 156.23, 134.11, 129.40, 114.89, 107.63, 78.94, 56.64, 49.22, 39.73, 38.38, 36.88, 29.46, 28.57, 28.23, 25.97, 24.64, 18.82, 18.16, 15.95, 3.74, 4.86. To a solution of compound 29 (5.0 g, 11.6 mmol) in CHCl3 (50 mL) was added BF3$Et2O (15 mL) at 0  C. After the addition was complete, the mixture was stirred for 1 h at room temperature and then poured into ice water (50 mL), extracted with AcOEt (30 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 1/1 v/v) to give compound 30 (2.89 g, 79%) as a white solid. 1H NMR (400 MHz, CDCl3) d 8.11 (s, 1 H), 7.03 (d, J ¼ 8.4 Hz, 1 H), 6.86 (d, J ¼ 2.6 Hz, 1 H), 6.73 (dd, J ¼ 8.4, 2.6 Hz, 1 H), 3.81 (s, 3 H), 3.01 (d, J ¼ 15.1 Hz, 1 H), 2.98e2.72 (m, 2 H), 2.51 (d, J ¼ 15.1 Hz, 1 H), 1.99e1.65 (m, 3 H), 1.39 (s, 3 H), 1.30 (s, 3 H), 1.21 (s, 3 H). To a solution of compound 30 (64 mg, 0.2 mmol) in EtOH (10 mL) was added H2SO4 (catalytic amount) and then the reaction mixture was heated to reflux for 2 h. After cooling, the reaction mixture was poured into saturated NaHCO3 (10 mL) and extracted with AcOEt (10 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated to give compound 31 (68 mg, 98%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) d 7.28 (s, 1 H), 6.91 (s, 1 H), 4.45 (d, J ¼ 5.0 Hz, 1 H), 4.20 (q, J ¼ 7.1 Hz, 2 H), 3.76 (s, 3 H), 3.09 (d, J ¼ 8.1 Hz, 1 H), 2.92e2.59 (m, 2 H), 2.32 (d, J ¼ 13.0 Hz, 1 H), 1.86e1.48 (m, 4 H), 1.41 (d, J ¼ 3.8 Hz, 1 H), 1.25 (t, J ¼ 7.1 Hz, 3 H), 1.18 (d, J ¼ 11.5 Hz, 1 H), 1.13 (s, 3 H), 0.98 (s, 3 H), 0.79 (s, 3 H).

6.9. Synthesis of compound 34 To a solution of compound 15 (895 mg, 2.5 mmol) in dry DCM (15 mL) was added m-CPBA (1 g, 5 mmol, 85% wt). The reaction mixture was stirred for 12 h at room temperature and then washed with aqueous NaOH (10 mL, 0.5 M), dried over Na2SO4 and concentrated to give crude product compound 32. To a solution of crude product compound 32 in MeOH (15 mL) was added NaOH (400 mg, 10 mmol). The reaction mixture was heated to reflux for 3 h and then concentrated. Hydrochloric acid (20 mL, 1 M) was added to the residue and extracted with EtOAc (10 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 2/1 v/v) to give compound 33 (520 mg, 72% over two steps) as a white solid. 1H NMR (400 MHz, CDCl3) d 6.71 (s, 1 H), 6.59 (s, 1 H), 3.84 (s, 3 H), 3.31 (dd, J ¼ 11.3, 4.8 Hz, 1 H), 2.94e2.63 (m, 2 H), 2.33e2.19 (m, 1 H),

17

1.93e1.49 (m, 6 H), 1.30 (dd, J ¼ 12.2, 1.7 Hz, 1 H), 1.19 (s, 3 H), 1.07 (s, 3 H), 0.89 (s, 3 H). To a solution of compound 33 (290 mg, 1 mmol) in DMF (8 mL) was added K2CO3 (276 mg, 2 mmol) and BnBr (342 mg, 2 mmol). The reaction mixture was stirred for 12 h at room temperature and then was added with water (15 mL) and extracted with EtOAc (10 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 5/1 v/v) to give compound 34 (293 mg, 77%) as a white solid. 1H NMR (400 MHz, CDCl3) d 7.44 (d, J ¼ 7.4 Hz, 2 H), 7.36 (t, J ¼ 7.5 Hz, 2 H), 7.30 (d, J ¼ 7.1 Hz, 1 H), 6.77 (s, 1 H), 6.57 (s, 1 H), 5.08 (s, 2 H), 3.85 (s, 3 H), 3.30 (dd, J ¼ 11.3, 4.8 Hz, 1 H), 2.89e2.65 (m, 2 H), 2.33e2.21 (m, 1 H), 1.92e1.66 (m, 4 H), 1.30 (d, J ¼ 10.7 Hz, 1 H), 1.19 (s, 3 H), 1.09e1.02 (m, 3 H), 0.89 (s, 3 H).

6.10. Synthesis of compounds 38 and 39 To a solution of compound 15 (1.79 g, 5 mmol) in MeOH (20 mL) was added NaOH (400 mg, 10 mmol). The reaction mixture was heated to reflux for 3 h and then concentrated. Water (10 mL) was added to the residue and extracted with EtOAc (10 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated to give compound 35 (1.44 g, 99%) as a white solid. To a solution of compound 35 (651 mg, 2.05 mmol) in MeOH (20 mL) was added NaBH4 (228 mg, 6 mmol). The reaction mixture was stirred for 3 h at room temperature and then concentrated. The residue was poured into water and extracted with EtOAc (10 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated to give the crude product compound 36, and used in the next step without purification. To a solution of 35 (1.1 g, 3.48 mmol) in dry THF (20 mL) was added CH3MgCl (5 mL, 3 M in THF). The reaction mixture was stirred for 6 h at room temperature and then poured into saturated NH4Cl (30 mL) and extracted with AcOEt (15 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated to give the crude product compound 37, and used in the next step without purification. To a solution of crude product compound 36 or compound 37 in dry THF (20 mL) was added TsOH (catalytic amount). The reaction mixture was heated to reflux for 5 h and then poured into half saturated NaHCO3 (20 mL) and extracted with AcOEt (15 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 3/1 v/v) to give compound 38 (465 mg, 75% over two steps) as a white solid. 1H NMR (400 MHz, CDCl3) d 7.13 (s, 1 H), 6.96 (dd, J ¼ 17.8, 11.2 Hz, 1 H), 6.73 (s, 1 H), 5.68 (d, J ¼ 17.7 Hz, 1 H), 5.20 (d, J ¼ 11.1 Hz, 1 H), 3.79 (d, J ¼ 13.2 Hz, 3 H), 3.31 (dd, J ¼ 11.1, 5.0 Hz, 1 H), 3.00e2.72 (m, 2 H), 2.37e2.22 (m, 1 H), 1.94e1.67 (m, 4 H), 1.32 (dd, J ¼ 12.1, 1.6 Hz, 1 H), 1.21 (s, 3 H), 1.08 (s, 3 H), 0.90 (s, 3 H); or compound 39 (776 mg, 71% over two steps) as a white solid. 1H NMR (400 MHz, CDCl3) d 6.87 (s, 1 H), 6.74 (s, 1 H), 5.08 (d, J ¼ 20.6 Hz, 2 H), 3.80 (s, 3 H), 3.31 (dd, J ¼ 11.0, 5.0 Hz, 1 H), 2.99e2.69 (m, 2 H), 2.30 (dt, J ¼ 12.8, 2.9 Hz, 1 H), 2.10 (s, 3 H), 1.96e1.68 (m, 4 H), 1.33 (dd, J ¼ 12.1, 1.6 Hz, 1 H), 1.23 (s, 3 H), 1.08 (s, 3 H), 0.91 (s, 3 H).

6.11. General procedure for the synthesis of compounds 40e49 Compounds 40e49 were prepared by a similar procedure (three steps: oxidation by IBX; Claisen condensation with ethyl formate; cyclization with hydrazine hydrate) described for compound 5.

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6.11.1. Compound 40 Light yellow solid; yield: 68% (over three steps); mp: 156e160  C. 1H NMR (500 MHz, DMSO-d6) d 12.43 (s, 1 H), 7.42 (s, 1 H), 7.29 (s, 1 H), 7.21 (s, 1 H), 3.91 (s, 3 H), 3.28 (s, 1 H), 2.89 (dd, J ¼ 16.6, 3.9 Hz, 1 H), 2.75e2.65 (m, 1 H), 2.35 (d, J ¼ 14.6 Hz, 1 H), 1.94e1.88 (m, 1 H), 1.76e1.51 (m, 2 H), 1.29 (s, 3 H), 1.19 (s, 3 H), 1.13 (s, 3 H); 13C NMR (100 MHz, DMSO-d6) d 158.93, 154.70, 133.57, 128.44, 116.56, 111.11, 109.61, 98.09, 56.18, 49.23, 34.60, 33.37, 31.01, 29.70, 23.91, 23.14, 18.95; HRMS(ESI): calcd for C20H24N3O [M þ H]þ; 322.1919, found 322.1938. 6.11.2. Compound 41 Light yellow solid; yield: 86% (over three steps); mp: 175e178  C. 1H NMR (400 MHz, CDCl3) d 7.42 (s, 1 H), 6.87 (s, 1 H), 6.82 (s, 1 H), 3.85 (d, J ¼ 6.2 Hz, 3 H), 3.16 (d, J ¼ 14.8 Hz, 1 H), 2.98e2.70 (m, 2 H), 2.70e2.44 (m, 3 H), 2.02e1.62 (m, 3 H), 1.42 (s, 3 H), 1.36e1.30 (m, 3 H), 1.24e1.19 (m; 6 H); 13C NMR (100 MHz, CDCl3) d 156.09, 150.51, 145.23, 133.40, 130.63, 129.25, 127.38, 113.27, 107.68, 55.62, 50.24, 39.47, 35.76, 33.94, 31.58, 30.84, 24.79, 23.63, 22.85, 20.16, 14.21; HRMS(ESI): calcd for C21H29N2O [M þ H]þ; 325.2280; found 325.2294. 6.11.3. Compound 42 Light yellow solid; yield: 48% (over three steps); mp: 177e181  C. 1H NMR (400 MHz, CDCl3) d 7.47 (s, 1 H), 6.85 (s, 1 H), 6.79 (s, 1 H), 3.83 (s, 3 H), 3.17 (d, J ¼ 14.9 Hz, 1 H), 2.94e2.71 (m, 2 H), 2.63e2.45 (m, 3 H), 1.95 (dd, J ¼ 12.5, 5.4 Hz, 1 H), 1.85 (d, J ¼ 12.5 Hz, 1 H), 1.79e1.67 (m, 1 H), 1.67e1.53 (m, 2 H), 1.46 (s, 2 H), 1.38 (s, 3 H), 1.20 (s, 3 H), 0.97 (t, J ¼ 7.3 Hz, 3 H); 13C NMR (100 MHz, CDCl3) d 156.17, 150.50, 145.24, 133.33, 130.11, 129.20, 127.23, 113.29, 107.72, 55.62, 50.20, 39.46, 35.74, 33.93, 32.01, 31.57, 30.80, 24.80, 23.62, 23.17, 20.15, 14.41; HRMS(ESI): calcd for C22H31N2O[M þ H]þ; 339.2431; found 339.2470. 6.11.4. Compound 43 Light yellow solid; yield: 54% (over three steps); mp: 157e159  C. 1H NMR (400 MHz, CDCl3) d 7.43 (s, 1 H), 6.84 (d, J ¼ 12.7 Hz, 2 H), 3.85 (s, 3 H), 3.17 (d, J ¼ 14.8 Hz, 1 H), 3.00e2.69 (m, 2 H), 2.66e2.46 (m, 3 H), 1.95 (dd, J ¼ 12.4, 5.1 Hz, 1 H), 1.89e1.66 (m, 2 H), 1.63e1.52 (m, 2 H), 1.43 (s, 3 H), 1.35 (s, 3 H), 1.22 (s, 3 H), 0.95 (t, J ¼ 7.3 Hz, 3 H); 13C NMR (100 MHz, CDCl3) d 156.04, 150.25, 145.11, 133.45, 129.90, 129.29, 127.15, 113.12, 107.63, 55.53, 50.11, 39.36, 35.65, 33.81, 32.17, 31.46, 30.71, 29.72, 29.46, 24.70, 23.50, 22.83, 20.05, 14.08; HRMS(ESI): calcd for C23H33N2O [M þ H]þ; 353.2587; found 353.2593. 6.11.5. Compound 44 Light yellow solid; yield: 54% (over three steps); mp: 127e131  C. 1H NMR (400 MHz, CDCl3) d 7.52 (s, 1 H), 7.44 (s, 1 H), 6.85 (s, 1 H), 6.80 (s, 1 H), 3.84 (d, J ¼ 6.4 Hz, 3 H), 3.16 (d, J ¼ 14.9 Hz, 1 H), 2.96e2.71 (m, 2 H), 2.67e2.34 (m, 3 H), 2.02e1.64 (m, 3 H), 1.57 (dt, J ¼ 15.1, 7.4 Hz, 2 H), 1.46e1.40 (m, 3 H), 1.35 (s, 3 H), 1.33e1.24 (m, 10 H), 1.21 (s, 4 H), 0.89 (t, J ¼ 6.7 Hz, 4 H); 13C NMR (100 MHz, CDCl3) d 156.14, 150.50, 145.19, 133.35, 129.99, 129.47, 127.26, 113.28, 107.73, 55.63, 50.21, 39.46, 35.75, 33.93, 32.07, 31.57, 30.81, 30.08, 29.92, 29.87, 29.65, 29.43, 24.80, 23.62, 22.83, 20.16, 14.26; HRMS(ESI): calcd for C27H41N2O [M þ H]þ; 409.3219; found 409.3210. 6.11.6. Compound 45 Light yellow solid; yield: 43% (over three steps); mp: 212e216  C. 1H NMR (400 MHz, CDCl3) d 7.60 (br.s, 1 H), 7.43 (s, 1 H), 7.27 (s, 1 H), 6.87 (s, 1 H), 3.91 (d, J ¼ 6.2 Hz, 3 H), 3.15 (d, J ¼ 14.8 Hz, 1 H), 2.96e2.69 (m, 2 H), 2.55 (d, J ¼ 14.7 Hz, 1 H), 1.94 (dd, J ¼ 12.2, 5.0 Hz, 1 H), 1.87e1.62 (m, 2 H), 1.42 (s, 3 H), 1.33 (s, 3 H), 1.19 (s,

3 H); 13C NMR (100 MHz, CDCl3) d 154.36, 150.52, 147.53, 133.32, 133.02, 129.70, 112.89, 109.79, 109.37, 56.49, 49.93, 39.65, 35.67, 33.96, 31.54, 30.51, 24.78, 23.61, 19.88; HRMS(ESI): calcd for C19H24BrN2O[M þ H]þ; 375.1072; found 375.1054. 6.11.7. Compound 46 Light yellow solid; yield: 74% (over three steps); mp: 114e117  C. 1 H NMR (400 MHz, CDCl3) d 7.53 (s, 1 H), 7.41 (s, 1 H), 6.95 (s, 1 H), 4.35 (q, J ¼ 7.1 Hz, 2 H), 3.91 (s, 3 H), 3.16 (d, J ¼ 14.7 Hz, 1 H), 3.03e2.70 (m, 2 H), 2.56 (d, J ¼ 14.7 Hz, 1 H), 1.96 (dd, J ¼ 12.6, 5.5 Hz, 1 H), 1.90e1.61 (m, 2 H), 1.41 (s, 3 H), 1.37 (t, J ¼ 7.1 Hz, 3 H), 1.33 (s, 3 H), 1.20 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 166.24, 157.64, 152.81, 150.40, 133.32, 132.13, 127.69, 118.45, 112.72, 109.85, 60.83, 56.34, 49.88, 40.01, 35.56, 33.98, 31.55, 30.58, 24.68, 23.66, 19.93, 14.47; HRMS(ESI): calcd for C22H29N2O3[M þ H]þ; 369.2178; found 369.2141. 6.11.8. Compound 47 Light yellow solid; yield: 85% (over three steps). 1H NMR (400 MHz, DMSO-d6) d 7.44e7.30 (m, 6 H), 6.93 (s, 1 H), 6.68 (s, 1 H), 5.01 (s, 2 H), 3.75 (s, 3 H), 3.17 (d, J ¼ 14.7 Hz, 1 H), 2.84e2.60 (m, 2 H), 2.30 (d, J ¼ 14.7 Hz, 1 H), 1.91e1.78 (m, 1 H), 1.68e1.56 (m, 2 H), 1.28 (s, 3 H), 1.18 (s, 3 H), 1.08 (s, 3 H). 6.11.9. Compound 48 Light yellow solid; yield: 45% (over three steps); mp: 106e109  C. 1H NMR (500 MHz, CDCl3) d 7.37 (s, 1 H), 7.18 (s, 1 H), 6.98 (dd, J ¼ 17.7, 11.1 Hz, 1 H), 6.85 (s, 1 H), 5.72 (d, J ¼ 17.7 Hz, 1 H), 5.23 (d, J ¼ 11.2 Hz, 1 H), 3.85 (d, J ¼ 10.8 Hz, 3 H), 3.15 (d, J ¼ 14.7 Hz, 1 H), 2.98e2.74 (m, 2 H), 2.55 (d, J ¼ 14.7 Hz, 1 H), 1.95 (dd, J ¼ 12.8, 5.6 Hz, 1 H), 1.86e1.68 (m, 2 H), 1.38 (s, 3 H), 1.30 (s, 3 H), 1.21 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 155.53, 150.59, 147.77, 133.23, 131.56, 127.78, 126.98, 125.02, 114.23, 113.15, 108.47, 55.86, 50.15, 39.69, 35.71, 33.97, 31.58, 30.84, 24.73, 23.65, 20.11; HRMS(ESI): calcd for C21H27N2O[M þ H]þ; 323.2123; found 323.2147. 6.11.10. Compound 49 Light yellow solid; yield: 75% (over three steps); mp: 104e107  C. 1H NMR (400 MHz, CDCl3) d 8.36 (br.s, 1 H), 7.43 (s, 1 H), 6.89 (d, J ¼ 25.5 Hz, 2 H), 5.11 (d, J ¼ 16.9 Hz, 2 H), 3.85 (s, 3 H), 3.17 (d, J ¼ 14.7 Hz, 1 H), 2.99e2.69 (m, 2 H), 2.58 (d, J ¼ 14.7 Hz, 1 H), 2.13 (s, 3 H), 1.96 (dd, J ¼ 12.3, 4.8 Hz, 1 H), 1.89e1.66 (m, 2 H), 1.43 (s, 3 H), 1.34 (s, 3 H), 1.23 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 155.35, 150.61, 146.94, 143.97, 133.22, 130.83, 129.69, 127.60, 115.04, 113.21, 108.57, 55.85, 50.16, 39.56, 35.69, 33.96, 31.58, 30.75, 24.76, 23.65, 23.40, 20.11. HRMS(ESI): calcd for C22H29N2O [M þ H]þ; calcd 337.228; found 337.2254. 6.12. Synthesis of compound 50 To a solution of compound 46 (90 mg, 0.24 mmol) in THF (10 mL) was added NaOH（50 mg, 1.25 mmol）at 0  C. The reaction mixture was heated to reflux for 3 h then poured into hydrochloric acid (20 mL, 0.1 M), extracted with AcOEt (10 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated to give compound 50 (78 mg, 90%) as a light yellow solid; mp: 262e263  C. 1H NMR (400 MHz, DMSO-d6) d 7.37 (s, 1 H), 7.32 (s, 1 H), 7.08 (s, 1 H), 3.82 (s, 3 H), 3.26 (d, J ¼ 14.8 Hz, 1 H), 2.87 (dd, J ¼ 16.2, 3.6 Hz, 1 H), 2.78e2.63 (m, 1 H), 2.36 (d, J ¼ 14.7 Hz, 1 H), 1.92e1.85 (m, 1 H), 1.75e1.53 (m, 2 H), 1.30 (s, 3 H), 1.20 (s, 3 H), 1.13 (s, 3 H); 13C NMR (100 MHz, DMSO-d6) d167.12, 156.69, 152.37, 148.51, 133.07, 131.22, 127.04, 118.84, 111.51, 109.96, 55.90, 49.52, 34.89, 33.43, 31.06, 30.46, 29.98, 24.15, 23.18, 21.10, 19.31; HRMS(ESI): calcd for C20H25N2O3 [M þ H]þ; 341.1865; found 341.1861.

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6.13. General procedure for the synthesis of compounds 51 and 52 To a solution of compound 47 or 49 (0.39 mmol) in MeOH (10 mL) was added 5% Pd on carbon (20 mg). The reaction mixture was subjected to 1 atm of H2 and stirred for 12 h at room temperature. Then the reaction mixture was filtered and the filtrate was concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 1/1 v/v) to obtain the desired compounds. 6.13.1. Compound 51 White solid; yield: 89%; mp: 262e263  C. 1H NMR (400 MHz, CDCl3) d 7.52 (s, 1 H), 6.80 (s, 1 H), 6.64 (s, 1 H), 3.90 (s, 3 H), 3.28e3.06 (m, 1 H), 2.96e2.70 (m, 2 H), 2.54 (d, J ¼ 14.9 Hz, 1 H), 1.99e1.64 (m, 3 H), 1.49 (s, 3 H), 1.41 (s, 3 H), 1.18 (s, 3 H); 13C NMR (100 MHz, DMSO-d6) d 146.25, 144.42, 137.52, 127.36, 114.79, 111.75, 109.83, 55.78, 49.99, 38.56, 35.50, 33.32, 31.10, 30.40, 24.45, 23.09, 19.53; HRMS(ESI): calcd for C19H25N2O2 [M þ H]þ; 313.1916; found 313.1951. 6.13.2. Compound 52 White solid; yield: 83%; mp: 123e126  C. 1H NMR (500 MHz, CDCl3) d 7.38 (s, 1 H), 6.90 (s, 1 H), 6.82 (s, 1 H), 3.84 (s, 3 H), 3.29e3.18 (m, 1 H), 3.14 (d, J ¼ 14.7 Hz, 1 H), 2.97e2.74 (m, 2 H), 2.56 (d, J ¼ 14.7 Hz, 1 H), 1.94 (dd, J ¼ 12.7, 5.7 Hz, 1 H), 1.84 (d, J ¼ 12.6 Hz, 1 H), 1.78e1.65 (m, 1 H), 1.38 (s, 3 H), 1.30 (s, 3 H), 1.24e1.17 (m, 9 H); 13C NMR (100 MHz, CDCl3) d 155.54, 150.57, 144.90, 135.01, 133.30, 127.39, 126.44, 113.34, 107.87, 55.71, 50.15, 39.41, 35.67, 33.93, 31.58, 30.95, 26.63, 24.79, 23.63, 22.95, 22.81, 20.18; HRMS(ESI): calcd for C22H31N2O[M þ H]þ; 339.2436; found 339.2436. 6.14. General procedure for the synthesis of compounds 53e55 To a solution of the methoxy derivatives (162 mg, 0.5 mmol) in DCM (10 mL) was added BBr3 (1.5 mL, 1 M in DCM) at 78  C. The reaction mixture was stirred for 2 h at 78  C and then poured into water (100 mL), extracted with DCM (10 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (DCM/MeOH, 30/1 v/v) to obtain the desired compounds. 6.14.1. Compound 53 Light yellow solid; yield: 84%; mp: 142e145  C. 1H NMR (400 MHz, CDCl3) d 7.56 (br.s, 2 H), 7.38 (s, 1 H), 6.86 (s, 1 H), 6.80 (s, 1 H), 3.04 (d, J ¼ 14.8 Hz, 1 H), 2.94e2.70 (m, 2 H), 2.62 (q, J ¼ 7.3 Hz, 2 H), 2.50 (d, J ¼ 14.8 Hz, 1 H), 1.98e1.63 (m, 3 H), 1.39 (s, 3 H), 1.30 (s, 3 H), 1.27e1.22 (m, 3 H), 1.14 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 152.35, 150.77, 145.60, 133.13, 129.43, 128.31, 127.40, 113.49, 112.48, 50.12, 39.09, 35.62, 33.92, 31.53, 30.82, 24.81, 23.60, 22.75, 20.19, 14.12; HRMS(ESI): calcd for C20H27N2O [M þ H]þ; 311.2118; found 311.2145. 6.14.2. Compound 54 Light yellow solid; yield: 75%; mp: 165e167  C. 1H NMR (500 MHz, CDCl3) d 7.36 (s, 1 H), 7.19 (s, 1 H), 7.03 (s, 1 H), 3.09 (d, J ¼ 14.8 Hz, 1 H), 2.94e2.71 (m, 2 H), 2.51 (d, J ¼ 14.7 Hz, 1 H), 1.93 (dd, J ¼ 12.7, 5.6 Hz, 1 H), 1.83e1.63 (m, 2 H), 1.38 (s, 3 H), 1.29 (s, 3 H), 1.17 (s, 3 H); 13C NMR (100 MHz, DMSO-d6) d 152.06, 147.62, 132.21, 127.83, 113.52, 111.22, 106.85, 49.41, 38.81, 35.31, 33.34, 31.05, 29.78, 24.56, 23.15, 19.36; HRMS(ESI): calcd for C18H22BrN2O [M þ H]þ; 361.0916; found 361.0946.

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6.14.3. Compound 55 Light yellow solid; yield: 57%; mp: 156e158  C. 1H NMR (400 MHz, CDCl3) d 7.70 (br.s, 2 H), 7.37 (s, 1 H), 6.89 (s, 1 H), 6.80 (s, 1 H), 3.35e3.11 (m, 1 H), 3.02 (d, J ¼ 14.8 Hz, 1 H), 2.96e2.68 (m, 2 H), 2.49 (d, J ¼ 14.7 Hz, 1 H), 2.00e1.59 (m, 3 H), 1.38 (s, 3 H), 1.31e1.20 (m, 9 H), 1.13 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 151.83, 150.72, 145.23, 133.24, 132.85, 127.33, 126.61, 113.46, 112.66, 50.09, 39.04, 35.57, 33.90, 31.54, 30.95, 26.94, 24.79, 23.59, 22.90, 22.77, 20.22; HRMS(ESI): calcd for C21H29N2O [M þ H]þ; 325.228; found 325.2272. 6.15. Synthesis of compounds 56 To a solution of 53 (62 mg, 0.2 mmol) and DMAP (6 mg, 0.05 mmol) in dry DCM (10 mL) was added Ac2O (102 mg, 1 mmol) under N2. The reaction mixture was stirred for 6 h at room temperature, and then concentrated. To the residue was added MeOH (10 mL) and TEA (50 mg, 0.5 mmol). The reaction mixture was stirred for another 2 h at room temperature and then concentrated. The residue was purified by silica gel chromatography (DCM/ MeOH, 50/1 v/v) to give 56 (54 mg, 77%) as a white solid; mp: 89e92  C. 1H NMR (400 MHz, CDCl3) d 7.54 (s, 1 H), 7.37 (s, 1 H), 6.98 (d, J ¼ 5.9 Hz, 2 H), 3.09 (d, J ¼ 14.8 Hz, 1 H), 3.02e2.76 (m, 2 H), 2.62e2.41 (m, 3 H), 2.32 (s, 3 H), 1.95 (dd, J ¼ 12.4, 5.5 Hz, 1 H), 1.88e1.66 (m, 2 H), 1.40 (s, 3 H), 1.32 (s, 3 H), 1.23e1.13 (m, 6 H); 13C NMR (100 MHz, CDCl3) d 169.96, 150.66, 147.41, 145.93, 133.48, 133.09, 132.89, 129.65, 119.57, 113.17, 49.86, 39.19, 35.56, 33.92, 31.52, 31.10, 24.90, 23.61, 22.82, 21.02, 19.94, 14.10; HRMS(ESI): calcd for C22H29N2O2 [M þ H]þ; 353.2229; found 353.2251. 6.16. Synthesis of compounds 57 To a solution of 53 (52 mg, 0.17 mmol) in dry DMF (5 mL) was added K2CO3 (231 mg, 1.7 mmol) and allyl bromide (203 mg, 1.7 mmol) under N2. The reaction mixture was stirred for 10 h at room temperature and then poured into water (15 mL), extracted with AcOEt (10 mL  3). The combined organic extract was washed with brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel chromatography (DCM/MeOH, 50/1 v/v) to give 57 (33 mg, 56%) as a white solid; mp: 183e186  C. 1H NMR (400 MHz, CDCl3) d 7.40 (s, 1 H), 6.88 (s, 1 H), 6.83 (s, 1 H), 6.23e5.98 (m, 1 H), 5.45 (dd, J ¼ 17.3, 1.5 Hz, 1 H), 5.29 (dd, J ¼ 10.5, 1.3 Hz, 1 H), 4.57 (d, J ¼ 5.0 Hz, 2 H), 3.12 (d, J ¼ 14.8 Hz, 1 H), 2.95e2.75 (m, 2 H), 2.68e2.60 (m, 2 H), 2.54 (d, J ¼ 14.7 Hz, 1 H), 1.94 (dd, J ¼ 12.5, 5.5 Hz, 1 H), 1.86e1.63 (m, 2 H), 1.40 (s, 3 H), 1.32 (s, 3 H), 1.25e1.16 (m, 6 H); 13C NMR (100 MHz, CDCl3) d 155.05, 150.46, 145.29, 134.07, 133.61, 130.96, 129.32, 127.64, 116.89, 113.13, 109.34, 69.19, 50.24, 39.43, 35.79, 33.92, 31.58, 30.87, 24.80, 23.64, 22.99, 20.18, 14.26; HRMS(ESI): calcd for C23H21N2O [M þ H]þ; 351.2436; found 351.2456. 6.17. Biological assay 6.17.1. In vitro antibacterial assay The strains used in this study were S. aureus Newman and five multidrug-resistant S. aureus (NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271). All strains were grown at 37  C overnight in TSB without the antibiotic. Overnight cultures diluted 1000 fold were grown at 37  C for 2e3 h until A600 0.6. Then bacteria were diluted 1:200 into fresh TSB medium, compounds were prepared in DMSO and diluted serially by two-fold to final concentrations in the range of 0.39e50 mg/mL. Equal volume of bacteria and compound were added to 96 well plates and mixed well by shaking. After 16h incubation, the A600 of each well was visualized by Bioteksynergy2. Experiments were performed three times for each condition.

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6.17.2. Cytotoxicity assay The cytotoxic activity against human fibroblast (HAF) cells in vitro was measured using the MTS assay. Stock solutions (25 mg/ mL) of the test compounds were dissolved in DMSO. Cells were cultured in a 96-well plate at a density of 1  105 cells and different concentrations of compounds were respectively added to each well. The incubation was permitted at 37  C, 5% CO2 atmosphere. After incubation for 24 h, the cells were treated with various concentrations of the compound for 24 h. 20 mL MTS reagent was added into each well of the 96-well assay plate containing the compounds in 100 mL of culture medium. The samples were incubated at 37  C, 5% CO2 atmosphere for 1e2 h. Response of HAF cells to the test compounds was determined spectrophotometrically at a single wavelength of 490 nm. The assay was measured three times, after which the average of IC50 was calculated. The cytotoxicity of each compound was expressed as the concentration of compound that reduced cell viability to 50% (IC50). 6.17.3. Morphological test The morphology of human adult fibroblast (HAF) cells was observed using cell HE staining assay. In brief, cells were cultured in a 12-well plate at the appropriate cell densities. After incubation for 24 h, cells were exposed to 1.56 mg/mL and 30 mg/mL concentrations of the compounds. After treatment for 24 h, the supernatant medium was removed and the cells were fixed by 95% alcohol. About 30 min later, the plate was washed by water for three times. After dying, the cell nucleuses were stained with 500 mL hematoxylin solution each well for 10 min. After staining, the hematoxylin was removed and the plate was washed by water for three times. After dying, the cells were differentiated with 500 mL 0.3% acid alcohol each well for several seconds until the background was almost colorless. After differentiation, the cells were immersed in 500 mL 0.5% dilute ammonia water for 10 min to make the cell nucleuses turn to blue. Then the plate was washed by water for three times and each well was added 500 mL eosin solutions for 5 min to stain the cytoplasm. After staining, the plate was washed by water for five times until the background was almost colorless. At last, the morphology was observed under a contrast microscope. Acknowledgments This work was supported by Shanghai Science and Technology Council (Grant 12ZR1408500), the Fundamental Research Funds for the Central Universities and and the National Science and Technology Major Project “Key New Drug Creation and Manufacturing Program” (2013ZX09507-004). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.11.015.

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