Design, synthesis and biological evaluation of novel quinoline [4,3-b]chromene derivatives as AChE inhibitors through an efficient one-pot, four-component microwave-mediated reaction           
Ming He1#, Baohua Xie1#, Pei He1, Haibing Zhou1*, Shengtang Huang2*, Chune Dong1*           
1. Hubei Province Key Laboratory of Allergy and Immunology,State Key Laboratory of Virology, Hubei Province Engineering and Technology Research Centre for Fluorinated Pharmaceuticals, Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China
2. School of Pharmacy, Hubei University of Science and Technology, Xianning 437100, China  
 
 
Abstract: An efficient synthesis of chromeno[4,3-b]quinoline derivatives via one-pot, four-component reaction of 4-hydroxycoumarin, formaldehyde, cyclohexanedione, ammonium ceric nitrate under microwave irradiation was accomplished. The structures of these compounds were unambiguously confirmed by single crystal X-ray diffraction. Furthermore, the anti-AChE activities of these compounds in vitro were investigated at concentrations of 20 μM and 50 μM by using a standard Ellman’s method. The relationship of inhibitory activities and structures of these chromeno [4,3-b]quinolines was also systematically studied. Of all the compounds investigated, 4ag emerged as the most potent AChE inhibitor with IC50 values of 5.63 µM, and it might be used as potent lead for the development anti-AChE agents. Moreover, molecular modelling was conducted to understand the optimal interaction of AChE with these types of compounds.   
Keywords: Chromeno [4,3-b]quinoline; Anti-AChE activities; Molecular modelling; Four-component reaction
CLC number: R916                Document code: A                 Article ID: 10031057(2018)1173518
 
 
1. Introduction
Coumarin-fused polycyclic heterocyclic molecules, in particular, chromeno [4,3-b]quinoline derivatives, are one type of the most important compounds that are present widely in nature, exhibiting interesting biological and pharmacological activities[1]. Many of these compoundsand related agents have antitumor[2], anti-HIV[3],antibacterial[4], antiviral[5], antifungal[6], anti-inflammatory[7] and anticoagulant activities[8] along with the properties of anti-neurodegenerative disorders, such as Alzheimer’s disease (AD)[9] and many more[10]. However, the method for simple syntheses of fused polycyclic heterocyclic derivatives is very limited. In particular, only few reports have studied the practical preparation of the chromeno[4,3-b]quinoline derivatives so far. The traditionalapproach for preparing chromeno[4,3-b]quinolines is through a multiple-step reaction of 4-hydroxycoumarin, aldehydes, ammonium acetate and 1,3-cyclohexadione (Scheme 1)[11]. In most cases, these reactions require long reaction time, large excess of ammonium acetate, and the stoichiometric amount of catalyst, or high reaction temperature, which does limit the scope and the application of the reaction. More recently, Farshid and coworkers have reported the synthesis of chromeno[4,3-b]quinolines through three-component one-pot reactionof 1,3-cyclohexadione, aldehydes and 4-aminocoumarin under microwave irradiation using heteropolyacid catalyst H3PW12O40[12]. Almost at the same time, Kumar has developed the Lewis acid Sc(OTF)3 catalyzed one-pot synthesis of chromeno[4,3-b]quinolines with good yields by using 4-aminocoumarin, aldehydes and dimedone[13].However, in these cases, 4-aminocoumarin is used as the starting material, and the harsh reaction conditions as well as special catalysts are required. Therefore, it is urgently necessary to synthesize the chromeno[4,3-b]quinoline derivatives through more practical methods.
 
  
Scheme 1. Methods for synthesis of chromeno[4,3-b]quinolones. 
 
As a progressive neurodegenerative disease, AD is increasingly threatening human health and life. It has received more and more attention in recent years. Donepezil, tacrine and galantamine, as acetylcholinesterase inhibitors, have already been approved for use in AD treatment[14].Despite the significant improvement has been achieved with AChE inhibitors, in the last two decades, no new types of AChE inhibitor have been approved by FDA, and most of them have failed in Phase II or Phase III clinical trials. Therefore, novel synthetic methodologies that can provide structurally diverse compounds which can effectively increase cholinergic neurotransmission are still highly desirable.As mentioned above, coumarin and chromene derivatives have been reported to have unique applications and advantages in the field of medicinal chemistry. However,reports on potent anti-AChE agents based on the chromeno[4,3-b]quinoline derivatives are very rare, and the method for facile synthesis of fused polycyclic heterocyclic derivatives is very limited.
Recently, the multicomponent reactions (MCRs) have emerged as attractive and powerful tools in modern organic synthesis, especially for heterocyclic molecules due to its efficiency, atom economy and structural complexity[15,16]. In view of our recent successful development of a series of heterocyclic compounds with potential biological activity[17,18], we aimed to design a simple procedure to prepare chromeno[4,3-b]quinoline derivatives, and evaluatetheir anti-AChE activities. To the best of our knowledge, this is the first time to accomplish the synthesis of chromeno[4,3-b]quinoline derivatives via four-component, one-pot reaction of 4-hydroxycoumarin,formaldehyde, cyclohexanedione, ammonium ceric nitrate under microwave irradiation. This work would open new prospects of the design and synthesis of highly specific anti-AChE agents.
Herein, we described a simple, convenient, straightforward synthesis of chromeno[4,3-b]quinoline derivatives as novel AChE inhibitors through four-component, one-pot multicomponent reactions under microwave conditions. A wide range of functional groups were tolerated in the developed protocol. The target molecules were obtained with high yields.Meanwhile, their anti-AChE activities were evaluated accordingly, and the preliminary structure-activityrelationships were also discussed. Finally, the possible interactions of these synthesized compounds wereanalyzed by both experimental approaches and molecular modeling. 
2. Results and discussion
In order to explore the reaction condition, ammonium acetate was used as the amine source in the reaction. Initially, the reaction of 4-hydroxycoumarin 1a with benzaldehyde 2a, 1,3-cyclohexadione 3a and ammonium acetate was used as a model reaction to examine the four-component. The mixture of 4-hydroxycoumarin 1a (1.0 equiv.), benzaldehyde 2a (1.0 equiv.),1,3-cyclohexadione3a (1.0 equiv.) and ammonium acetate (2.0 equiv.) was stirred in anhydrous acetic acid solution (3.0 mL) at 60 ºC for 1 h. However, no product was formed under these conditions, or at higher reaction temperature (100 ºC) and extended reaction time (2 h). Its of note that the reaction proceeded smoothly under microwave irradiation at 60 ºC for 0.5 h, providing the desired product 4a in 30% yield. Furthermore, the reaction conditions were optimized as shown in Table 1. In the beginning, different ammoniums were used as the amine sources (entries 15). When ammonium cerium nitrate (CAN) was applied in this reaction, 4a was afforded in 58% yield (entry 5). Since each CAN molecule contains two ammonium groups; so the amount of CAN was reduced to 0.5 equiv. It was pleased to see that the desired product 4a was obtained in 77% yield (entry 7). Therefore, 0.5 equiv. of CANwere used as the best amine source in the following reactions.  
 
Table 1. Four-component one-pot microwave reactions for synthesis of chromeno[4,3-b]quinoline derivativesa. 
 
aUnless otherwise specified, the reaction was carried out with 1a (0.5 mmol), 2a (0.5 mmol) and 3a (0.5 mmol) in the presence of amine source in solvent (3.0 mL) at 60 ºC for 30 min.  bIsolated yield.  
 
Subsequently, various solvents were extensively evaluated to determine the effects of the solvents on the reaction. Interestingly, the solvents played a crucial role in the reactivity of this multicomponent reaction (entries 8–17). Of all the solvents tested, including protic solvents and aprotic solvents, such as water, methanol, ethanol, formic acid, acetic acid, acetonitrile, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF), formic acid gave the best results (entry 8). It has been reported that the activity of CAN can be significantly improved in the mixed solvents[19]. Therefore, the effects of mixed solventswere also evaluated in this model reaction (entries 18–23). The results clearly showed that 1:1 (v/v) mixture of formic acid and water was the best solvent for this reaction, and the desired product 4a was formed in 93% yield (entry 19).
In order to find the optimum reaction time, the experiments were conducted in 1:1 (v/v) formic acid/water under microwave irradiation with 10, 20, 30 and 40 min, and the desired product 4a was obtained in yields of 70%, 83%, 93% and 47%, respectively (Table 2, entries 1–4). Accordingly, the best reaction time forthis reaction was 30 min. Finally, the reaction was performed at different irradiation power and different temperatures. The results showed that the best reaction power and temperature was 500 W and 60 ºC, respectively (Table 2, entries 3 and 5–9). On the basis of all of these experiments, the optimum reaction conditions were identified as 1:1 (v/v) formic acid/water at 60 ºC for 30 min under microwave irradiation in the presence of 0.5 equiv. of CAN. 
 
Table 2. Four-component one-pot microwave reactions for synthesis of chromeno[4,3-b]quinoline derivativesa. 
 
aUnless otherwise specified, the reaction was carried out with 1a (0.5 mmol), 2a (0.5 mmol) and 3a (0.5 mmol) in the presence of CAN (0.25 mmol) in H2O + HCO2H (1:1, v/v, 3.0 mL). bIsolated yield. 
 
With the optimized conditions in hand, we next explored the reaction scope by varying formaldehyde, cyclohexanedione, and the results were summarizedin Table 3. We were pleased to find that the reaction proceeded very well in all cases. The four-component, one-pot microwave reactions of 4-hydroxycoumarinwith a series of mono-substituted benzaldehydes bearingeither electron-donating or electron-withdrawing groups delivered the desired products 4a4q in 53%–99% yields. The structure of product 4e was unambiguously confirmed by single-crystal X-ray diffraction.  

Table 3. Scope of substituted benzaldehydes for synthesis of chromeno[4,3-b]quinoline derivativesa,b. 

 
aUnless otherwise specified, the reaction was carried out with 1a (0.5 mmol), 2 (0.5 mmol) and 3 (0.5 mmol) in the presence of CAN (0.25 mmol) in H2O + HCO2H (1:1, v/v, 3.0 mL). bIsolated yield. 
 
Multi-substituted benzaldehydes also exhibited good activities, affording the corresponding products4r4z in 50%–95% yields. In terms of formaldehyde,the substrates containing an alkyl chain (4aa), furanyl(4ab), thiopheneyl (4ac, 4ad), cyclic alkyl (4ae), methylenedioxybenzene (4af, 4ag) and naphthalene (4ah, 4ai, 4aj) groups all showed good activities, providing the desired products in excellent yield (86%–97%). Unfortunately, formaldehydes which contained N-heterocyclic substituents, such as pyrrole, piperidine, pyridine, indole and indazole, did not work at all. Finally, we used 5,5-dimethyl-1,3-cyclohexanedione in place of cyclohexanedioneto obtain three compounds smoothly (4ak, 4al, 4am),and the yield of the desired products was also excellent (85%, 88%, 98%). This fact, together with its compatibility with a wide range of functionalgroups, such as different substituted benzaldehydes, alkyl chain, furanyl, thiopheneyl, cyclic alkyl, methylenedioxybenzene and naphthalene that were observed in varying formaldehydes, made the new method a reliable and attractive approach for assembling chromeno[4,3-b]quinoline derivatives.
Subsequently, the synthesized compounds 4a4z and 4aa4am were evaluated for their anti-AChE activities in vitro at concentrations of 20 μM and 50 μM by using a standard Ellman’s method (Table 4)[20]. 
  
Table 4. The inhibition of anti-AChE activity of compounds 4 at 20 μM and 50 μM.  
  
aData are represented as inhibition% at 20 μM. bData are represented as inhibition% at 50 μM. All experiments were independently carried out at least three times. 
 
 
The results showed that all of the chromeno[4,3-b]quinoline compounds displayed moderate to excellent inhibitory activity at 20 μM and 50 μM. Especially, compounds 4a, 4e, 4ag, 4ak and 4am, exhibited excellent inhibitory activities (entries 1, 5, 33, 35 and 39). However, poor inhibitory activities were observed for 4p, 4x, 4aa, 4ai and 4aj (entries 16, 24, 27, 35 and 36). In order to discuss the structure-activity relationship of the compounds more intuitively, we analyzed the relationship of inhibitory activities and structures of the compounds, and the results were shown in Figure 1. Interestingly, when the substitution occurred at the para-position of the phenyl ring, the compounds displayed good inhibitory activities. In contrast, the results revealed that poor activity was observed when the substitution occurred at other position of the phenyl ring (Fig. 1A). Moreover, it was also apparent that compounds derived fromsteric hindered aldehydes led to decreased inhibition as compared with the less hindered analogues (Fig. 1B).
 
 
 
Figure 1. The inhibition of anti-AchE activity of compounds 4 bearing substituted phenyl group (A) and heterocycle group (B) at 20 μM and 50 μM.
 
 
Based on the results mentioned above, we chose the compounds with inhibitions of more than 50%at 50 μM toconduct IC50 assay further. Moreover, reference drug donepezil was tested for comparison. The results were shown in Table 5. In general, mostof compounds showed good inhibition potency. Especially, compounds 4ag and 4am exhibitednice inhibition activity against AChE with IC50 values of 5.63 µM and 6.57 µM, respectively (entries 13 and 16). These results were quite promising for further studies to discover novel anti-AChE agents for the treatment of AD.
To further understand the structure-binding relationships,the binding modes were investigated. The binding modes of 4ag and donepezil were studied by docking into the acetylcholinesterase. The docking experiments were carried out based on the published crystal structure of the acetylcholinesterase-related protein (PDB: 1W75) by using AutoDockTool (ADT) 4.2 (Fig. 2)[21]. The result showed that donepezil and 4ag both could well fit into the transfusion cavity of acetylcholinesterase-related protein. However, since donepezil has a longer diameter than 4ag, the former could bind to Ile287 and form a hydrogen bond with the length of 3.091 Å. Because the lengthof 4ag is shorter, hydrogen bond could not be formed with Ile287. However, 4ag can form hydrogen bonds with Ser200 and His400, and the lengths of the hydrogen bond were 2.032 Å and 2.475 Å, respectively. At the same time, from the docking results we can also see that when the substituent occurred in the para-position of the phenyl ring, the interactions with Ser200 and His400 residues were much stronger than otherpositions, which might be the reason that the compounds bearing substituents at the para-position of phenyl ring displayed much better anti-AchE activities.  
 

Table 5. The IC50 (μM) of anti-AchE activity of representative compounds 4. 

 
aData are represented as mean±SD. All experiments were independently carried out at least three times.
 
 
 
 
Figure 2. Computer modelling of the complex structures of the AChE protein with donepezil (A) and 4ag (B).  
 
3. Conclusions
In summary, we developed an efficient four-component one-pot microwave reaction for synthesis of chromeno[4,3-b]quinoline derivatives. These products were evaluated as potential AChE inhibitors and they exhibited moderate to excellent inhibitory activities. Of all the compounds investigated, compound 4ag emerged as the most potent anti-AChE inhibitor and might be used as potent lead for the development anti-AChE agents. The detailed SAR revealed that the substituent had great effect on the bioactivity. Further optimization on the phenyl ring of coumarinmight lead to high inhibitory activities. Further in-depth mechanism research of thesechromeno[4,3-b]quinolines is currently underway in our laboratory and will be reported in due course.
4. Experimental section
4.1. Instruments and reagents
Analytical-grade solvents were purchased and used without further purification. NMR spectra were recorded at 400 MHz for 1H spectra and 100 MHz for 13C spectra and calibrated from residual solvent signals. Chemical shifts were recorded in ppm (δ), and the residual solvent peak was used as an internal reference: proton (DMSO-d6, δ = 2.50, H2O δ = 3.33), carbon (DMSO-d6, δ = 39.52). Analytical thin-layer chromatography (TLC) was performed on silica gel aluminum sheets with an F-254 indicator. Visualization was accomplished by UV light. Mass spectra (MS) were recorded on an IonSpec 4.7 Tesla FTMS using the DART Positive. Single-crystal X-ray-diffraction measurements were carried out on a Bruker Smart-APEX-CCD diffract meter. All microwave irradiation experiments were carried out using the microwave oven XH-100B from Xianghu Company, China. The temperature in the MW experiments was measured by an eternal IR sensor. Purification by chromatography was performed using 230–400 mesh SiO2 with compressed air as a source of positive pressure.The following abbreviations were used for elution solvents: PE = petroleum ether, EA = ethyl acetate. All commercially available compounds were used as provided without further purification. The H2O used in the experiment was ultra-pure water, which was prepared by ultra-pure water system from Heal Force.
4.2. Typical procedure for the synthesis of chromeno[4,3-b]quinoline derivatives 4
A mixture contained well powdered 4-hydroxycoumarin1 (1.0 equiv., 81.1 mg, 0.5 mmol), 1,3-cyclohexadione 2 (1.0 equiv., 0.5 mmol), formaldehyde 3 (1.0 equiv., 0.5 mmol) and CAN (0.5 equiv., 137.1 mg, 0.25 mmol), and the solvent HCO2H+H2O (3 mL, v/v, 1:1) was added under the protection of argon. The resulting mixture was stirred at room temperature for 5 min, and then the mixture was transferred into the microwave vial. The vial was sealed, and the irradiation power was set at 500 W and irradiated in the microwave reactor at 60 ºC for 30 min. When the vial was removed from the apparatus, the solvent was evaporated under vacuum. The residue was added into 5 mL of water and extracted with ethyl acetate (3×20 mL). Then the organic layer was washed with brine and dried with anhydrous sodium sulfate. Evaporation of ethyl acetate gave a pale to yellow residue, which was purified by column chromatography to afford the pure product.
4a: The product was purified with silica gel chromatography (Petroleum etherEthyl acetate, 1:2, v/v), white solid, mp 325327 ºC; 1H NMR (400 MHz, DMSO-d6) δ: 9.79 (s, 1H), 8.33 (d, J = 7.3 Hz, 1H), 7.64 (t, 1H), 7.507.33 (m, J = 10.5 Hz, 2H), 7.307.00 (m, J= 11.1 Hz, 5H), 5.00 (s, 1H), 2.902.65 (m, 2H), 2.362.22 (m, 2H), 2.041.83 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.44, 160.85, 157.51, 152.46, 152.02, 147.66, 142.49, 132.37, 129.42, 124.47, 123.34, 118.78, 117.33, 115.14, 113.66, 113.53, 112.35, 102.21, 37.18, 34.25, 26.85, 21.18. HRMS: m/z calcd for C22H17NO3 [M+Na]+: 366.1101, found: 366.1107.
4b: The product was purified with silica gel chromatography (Petroleum etherEthyl acetate, 1:2, v/v), yellow solid, mp 274–276 °C;1H NMR (400 MHz,  DMSO-d6) δ: 9.74 (s, 1H), 8.35–8.30 (d, J = 8.0 Hz, 1H), 7.687.60 (t, J = 7.8 Hz, 1H), 7.51–7.41 (t, J = 7.3 Hz, 1H), 7.40–7.33 (d, J = 8.2 Hz, 1H), 7.14 (d, J = 8.7 Hz, 2H), 6.76 (d, J = 8.7 Hz, 2H), 4.93 (s, 1H), 3.66 (s, 3H), 2.90–2.65 (m, 2H), 2.38–2.20 (m, 2H), 2.08–1.90 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.44, 172.53, 160.83, 158.15, 152.47, 151.85, 142.25, 138.73,132.28, 129.12, 124.44, 123.39, 117.32, 113.87, 113.55,112.57, 102.55, 55.39, 37.17, 33.69, 26.83, 21.57, 21.24. HRMS: m/z calcd for C23H19NO4 [M+Na]+: 396.1206, found: 396.1210.
4c: The product was purified with silica gel chromatography (Petroleum etherEthyl acetate, 1:2, v/v), yellow solid, mp 345–347 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.79 (s, 1H), 8.35–8.31 (d, J = 7.4 Hz, 1H), 7.68–7.61 (t, J = 8.3 Hz, 1H), 7.48–7.42 (t, J = 7.4 Hz,1H), 7.40–7.36 (d, J = 8.2 Hz, 1H), 7.29–7.23 (m, J = 8.6 Hz, 2H), 7.07–6.99 (t, J = 8.9 Hz, 2H), 4.99 (s, 1H), 2.90–2.64 (m, 2H), 2.40–2.21 (m, 2H), 2.05–1.83 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.46, 160.80, 159.98, 152.52, 152.21, 142.56, 132.47, 130.03,129.93, 124.49, 123.48, 117.36, 115.26, 115.05, 113.46, 112.24, 102.10, 37.10, 34.10, 26.83, 22.98, 21.19. HRMS: m/z calcd for C22H16FNO3 [M+Na]+: 384.1006, found: 384.1010.
4d: The product was purified with silica gel chromatography (Petroleum etherEthyl acetate, 1:2, v/v), brown solid, mp 206208 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.82 (s, 1H), 8.34–8.30 (d, J = 7.8 Hz, 1H), 7.68–7.60 (t, J = 7.7 Hz, 1H), 7.48–7.42 (t, J = 7.5 Hz, 1H), 7.38–7.35 (d, J = 8.2 Hz, 1H), 7.30–7.25 (d, 2H), 7.23–7.18 (d, J = 8.4 Hz, 2H), 4.98 (s, 1H), 2.85–2.60 (m, 2H), 2.35–2.20 (m, 2H), 2.05–1.80 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.48, 171.94, 160.81, 152.51,152.22, 142.57, 132.48, 130.00, 129.92, 124.50, 123.47, 117.36, 115.26, 115.05, 113.45, 112.24, 102.10, 37.10, 34.10, 26.83, 22.98, 21.18. HRMS: m/z calcd for C22H16ClNO3 [M+Na]+: 400.0711, found: 400.0716.
4e: The product was purified with silica gel chromatography (Petroleum etherEthyl acetate, 1:2, v/v), yellow solid, mp 328–330 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.96 (s, 1H), 8.38–8.31 (d, J = 7.9 Hz, 1H), 7.68–7.60 (t, J = 7.7 Hz, 1H), 7.48–7.30 (m, J = 9.9 Hz, 4H), 7.24–7.14 (d, J = 8.4 Hz, 2H), 4.96 (s, 1H), 2.90–2.63 (m, 2H), 2.36–2.22 (m, 2H), 2.04–1.82 (m, 4.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.39,160.83, 152.57, 145.85, 132.47, 131.34, 130.49, 124.47, 123.63, 119.73, 117.33, 113.63, 111.89, 101.63, 37.11, 34.53, 26.98, 21.20. HRMS: m/z calcd for C22H16BrNO3 [M+Na]+: 444.0206, found: 444.0211.
4f: The product was purified with silica gel chromatography (Petroleum etherEthyl acetate, 1:2, v/v), yellow solid, mp 261–263 °C; 1H NMR (400 MHz,DMSO-d6) δ: 9.90 (s, 1H), 8.42–8.32 (d, J = 7.9 Hz, 1H), 8.16–8.04 (d, J = 8.6 Hz, 2H), 7.70–7.64 (t, J = 7.2 Hz, 1H), 7.60–7.44 (m, 3H), 7.44–7.35 (d, J = 8.2 Hz, 1H), 5.11 (s, 1H), 2.90–2.66 (m, 2H), 2.40–2.20 (m, 2H), 2.05–1.80 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.43, 160.72, 153.63, 152.82, 152.63, 146.43, 143.10,132.75, 129.63, 124.58, 123.79, 123.63, 117.44, 113.32, 111.46, 101.14, 37.00, 35.47, 26.88, 21.12, 14.55. HRMS: m/z calcd for C22H16N2O5 [M+Na]+: 411.0951, found: 411.0954.
4g: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), yellow solid, mp 305–307 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.74 (s, 1H), 8.38–8.25 (d, J = 7.8 Hz, 1H), 7.66–7.58 (t, J = 7.5 Hz, 1H), 7.48–7.40 (t, J = 7.5 Hz,1H), 7.39–7.35 (d, J = 8.2 Hz, 1H), 7.16–7.08 (d, J = 8.0 Hz, 2H), 7.05–6.95 (d, J = 7.9 Hz, 2H), 4.95 (s, 1H), 2.90–2.62 (m, 2H), 2.38–2.24 (m, 2H), 2.18 (s, 3H), 2.04–1.80 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.41, 160.81, 152.49, 151.95, 143.54, 142.39, 135.68, 132.36, 129.06, 128.03, 124.45, 123.40, 117.32, 113.53, 112.49, 102.41, 37.16, 34.16, 26.84, 21.22, 21.04. HRMS: m/z calcd for C23H19NO3 [M+Na]+: 380.1257, found: 380.1260.
4h: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:3, v/v), white solid, mp 338–340 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.70 (s, 1H), 9.17 (s, 1H), 8.35–8.25 (d, J = 7.8 Hz, 1H), 7.65–7.58 (t, J = 7.8 Hz, 1H), 7.46–7.40 (t, J = 7.6 Hz, 1H), 7.39–7.33 (d, J = 8.1 Hz, 1H), 7.07–6.98 (d, J = 8.5 Hz, 2H), 6.64–6.55 (d, J = 8.5 Hz, 2H), 4.89 (s, 1H), 2.88–2.62 (m, 2H), 2.38–2.22 (m, 2H), 2.06–1.80 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.46, 160.86, 156.22, 152.43, 151.72, 142.11, 137.09, 132.22, 129.06, 124.39, 123.33, 117.28, 115.20, 113.59, 112.70, 102.74, 37.21, 33.53, 26.84, 21.24. HRMS: m/z calcd for C22H17NO4 [M+Na]+: 382.1050, found: 382.1053.
4i: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), yellow solid, mp 318–320 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.92 (s, 1H), 8.39 (d, J = 7.8 Hz, 1H), 7.73–7.56 (m, 4H), 7.46 (t, J = 8.1 Hz, 3H), 5.07 (s, 1H), 2.93–2.70 (m, 2H), 2.29 (m, 2H), 2.02 (m,2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.41, 160.81, 152.48, 151.95, 143.55, 142.38, 135.68, 132.35, 129.06, 128.04, 124.44, 123.39, 117.32, 113.52, 112.48, 102.41, 37.16, 34.16, 26.83, 21.22, 21.04. HRMS: m/z calcd for C23H16F3NO3 [M+Na]+: 434.0974, found: 434.0982.
4j: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2,v/v), yellow solid, mp 345–347 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.76 (s, 1H), 8.36–8.28 (d, J = 7.7 Hz, 1H), 7.68–7.60 (t, J = 7.6 Hz, 1H), 7.48–7.41 (t, J = 7.6 Hz, 1H), 7.40–7.36 (d, J = 8.2 Hz, 1H), 7.14–6.98 (m,J = 7.6 Hz, 3H), 6.94–6.88 (d, J = 7.3 Hz, 1H), 4.97 (s, 1H), 2.92–2.64 (m, 2H), 2.34–2.26 (m, 2H), 2.22 (s, 3H), 2.051.80 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.42, 160.81, 152.49, 152.08, 146.37, 142.48, 137.33, 132.39, 128.78, 128.48, 127.39, 125.26, 124.47, 123.42, 117.34, 113.53, 112.39, 102.32, 37.16, 34.51, 26.86, 21.61, 21.20. HRMS: m/z calcd for C23H19NO3 [M+Na]+: 380.1257, found: 380.1261.
4k: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:3, v/v), whitesolid, mp 387–389 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.76 (s, 1H), 9.20 (s, 1H), 8.36–8.28 (d, J = 7.8 Hz, 1H), 7.68–7.58 (t, J = 7.4 Hz, 1H), 7.50–7.41 (t, J = 7.5 Hz, 1H), 7.41–7.34 (d, J = 8.2 Hz, 1H), 7.05–6.90 (t, J = 7.8 Hz, 1H), 6.78–6.58 (d, J = 7.5 Hz, 2H), 6.55–6.45 (d, J = 8.9 Hz, 1H), 4.94 (s, 1H),2.94–2.64 (m, 2H), 2.42–2.22 (m, 2H), 2.07–1.80 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.44, 160.85, 157.51, 152.46, 152.02, 147.66, 142.49, 132.37, 129.42, 124.47, 123.34, 118.78, 117.33, 115.14, 113.66, 113.53, 112.35, 102.21, 37.18, 34.25, 26.85, 21.18. HRMS: m/z calcd for C22H17NO4 [M+Na]+: 382.1050, found: 382.1053.
4l: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), yellow solid, mp 286–288 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.73 (s, 1H), 8.31 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 4.94 (s, 1H), 3.66 (s, 3H), 2.84 (d, J = 7.5 Hz, 1H), 2.70 (d, J = 10.6 Hz, 1H), 2.36–2.23 (m, 2H), 1.99 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.44, 172.53, 160.83, 158.15, 152.47, 151.83, 142.23, 138.74, 132.30, 129.12, 124.42, 123.37, 117.30, 113.87, 113.54, 112.58, 102.56, 55.38, 37.17, 33.70, 26.83, 21.55, 21.24. HRMS: m/z calcd for C23H19NO4 [M+Na]+: 396.1206, found: 396.1239. 
4m: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), brown solid, mp 292–294 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.85 (s, 1H), 8.35 (d, J = 7.3 Hz, 1H), 7.65 (d, J = 7.3 Hz, 1H), 7.58 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.0 Hz, 3H), 7.40 (s, 1H), 5.07 (s, 1H), 2.79 (m, 2H), 2.29 (m, 2H), 1.85 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.43, 160.72, 153.63, 152.83, 152.63, 146.43, 143.10, 132.74, 129.63, 127.81, 124.58, 123.79, 123.64, 117.43, 113.32, 111.46, 101.14, 72.68, 60.23, 37.00, 35.47, 26.87, 23.22, 21.23, 21.12, 14.55. HRMS: m/z calcd for C22H16N2O5 [M+Na]+: 411.0951, found: 411.0943.
4n: The product was purified with silica gelchromatography (Petroleum ether–Ethyl acetate,1:2, v/v), yellow solid, mp 235–237 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.78 (s, 1H), 8.34 (d, J = 6.5 Hz, 1H), 7.64 (d, J = 7.3 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.45–7.40 (d, J = 8.0 Hz, 1H), 7.38–7.20 (m, 3H), 7.15–7.10 (d, J = 6.2 Hz, 1H), 5.00 (s, 1H), 2.80 (m, 2H), 2.26 (m, 2H), 1.90 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.01, 160.45, 160.20, 152.01, 151.91, 147.93, 142.16, 131.99, 124.03, 123.00, 116.88, 113.06, 111.49, 106.40, 106.09, 101.47, 97.30, 36.72, 33.92, 26.40, 20.81. HRMS: m/z calcd for C23H16N2O3 [M+Na]+: 391.1053, found: 391.1074.
4o: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), white solid, mp 355–357 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.70 (s, 1H), 8.38–8.24 (d, J = 7.4 Hz, 1H), 7.66–7.56 (t, J = 8.3 Hz, 1H), 7.47–7.39 (t, J = 7.4 Hz, 1H), 7.38–7.32 (d, J = 8.2 Hz, 1H), 7.28–7.22 (d, J = 9.1 Hz, 1H), 7.13–7.03 (t, J = 8.6 Hz, 1H), 6.91–6.84 (d, J = 7.9 Hz, 1H), 6.83–6.74 (t, J = 7.4 Hz, 1H), 5.07 (s, 1H), 3.64 (s, 3H), 2.84–2.59 (m, 2H), 2.31–2.12 (m, 2H), 2.04–1.72 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.22, 160.56, 158.68, 152.47, 152.34, 142.91, 133.61, 132.13, 132.04, 127.99, 124.34, 123.22, 120.18, 117.20, 113.55, 112.68, 111.08, 100.97, 56.16, 37.25, 33.25, 26.88, 21.24. HRMS: m/z calcd for C23H19NO4 [M+Na]+: 396.1206, found: 396.1210.
4p: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), yellow solid, mp 386–388 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.84 (s, 1H), 8.36 (d, J = 7.7 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.50–7.30 (m, 4H), 7.20 (d, J = 8.3 Hz, 2H), 4.97 (s, 1H), 2.84 (d, J = 7.8 Hz, 1H), 2.70 (d, J = 13.5 Hz, 1H), 2.29 (m, 2H), 2.01 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.04, 171.51, 160.38, 152.08, 151.79, 142.13, 132.05, 129.57, 129.49,124.06, 123.04, 116.93, 114.83, 114.62, 113.02, 111.81, 101.67, 36.66, 33.66, 26.39, 22.54, 20.75. HRMS: m/z calcd for C22H16BrNO3 [M+Na]+: 444.0206, found: 444.0211.
4q: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate,1:2, v/v), brown solid, mp 194–196 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.74 (s, 1H), 8.32 (d, J = 7.8 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 7.12 (d, J = 7.9 Hz, 2H), 7.00 (d, J = 7.9 Hz, 2H), 4.96 (s, 1H), 2.84 (d, J = 7.6 Hz, 1H), 2.69 (d, J = 13.1 Hz, 1H), 2.342.24 (m, 2H), 2.19 (s, 3H), 2.00 (d, J = 13.0 Hz, 1H), 1.86 (d, J = 10.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ: 195.41, 160.81, 152.49, 151.95, 143.55, 142.39, 135.68, 132.36, 129.06, 128.04, 124.45, 123.40, 117.32, 113.52, 112.49, 102.41, 37.16, 34.16, 26.84, 21.22, 21.04. HRMS: m/z calcd for C23H19NO3 [M+Na]+: 380.1257, found: 380.1258.
4r: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:3, v/v), yellow solid, mp 360–362 °C; 1H NMR (400 MHz, DMSO-d6) δ: 10.09 (s, 1H), 9.71 (s, 1H), 8.45–8.30 (d, J = 7.8 Hz, 1H), 7.73–7.60 (t, J = 7.4 Hz, 1H), 7.53–743 (t, J = 7.5 Hz, 1H), 7.42–7.35 (d, J = 8.2 Hz, 1H), 6.97–6.88 (m, 1H), 6.82–6.73 (d, J = 7.7 Hz, 1H), 6.72–6.63 (m, J = 7.9 Hz, 1H), 5.08 (s, 1H), 2.89–2.63 (m, 2H), 2.42–2.24 (m, 2H), 2.05–1.75 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 197.62, 160.97, 153.69, 152.55, 151.30, 143.79, 142.55, 142.42, 136.34, 132.50, 125.02, 124.54, 123.63, 119.87, 117.35, 114.42,111.44, 101.67, 36.71, 30.20, 27.33, 20.95. HRMS: m/z calcd for C22H16FNO4 [M+Na]+: 400.0956, found: 400.0959.
4s: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), brown solid, mp 292–294 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.81 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.45 (t, J = 6.4 Hz, 1H), 7.38 (s, 1H), 7.02 (t, J = 9.4 Hz, 1H), 6.72 (s, 2H), 5.06 (s, 1H), 2.82–2.65 (m, 2H), 2.30–2.23 (m, 2H), 1.95 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.31, 160.47,152.76, 152.61, 143.07, 135.54, 135.39, 133.97, 132.68, 124.54, 123.57, 118.22, 117.97, 117.40, 116.01, 113.18,110.76, 100.45, 36.98, 31.42, 26.80, 21.19. HRMS: m/z calcd for C22H15BrFNO3 [M+Na]+: 462.0112, found: 462.0120.
4t: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2,v/v), yellow solid, mp 270–272 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.79 (s, 1H), 8.34 (d, J = 7.7 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.45 (t, J = 7.4 Hz, 1H), 7.37 (d, J = 8.0 Hz, 3H), 7.27 (d, J = 6.3 Hz, 1H), 5.25 (s, 1H), 2.80 (d, J = 7.6 Hz, 1H), 2.70 (d, J = 9.6 Hz, 1H), 2.24 (m, 2H), 1.98 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.30, 171.77, 160.64, 152.34, 152.05, 142.39, 132.31, 129.83, 129.75, 124.32, 123.30, 117.19, 115.09, 114.88, 113.28, 112.07, 101.93, 36.92, 33.92, 26.65, 22.80, 21.01. HRMS: m/z calcd for C22H15Cl2NO3 [M+Na]+: 434.0321, found: 434.0327.
4u: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), white solid, mp 348–350 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.77 (s, 1H), 8.37–8.29 (d, J = 7.8 Hz, 1H), 7.66–7.58 (t, J = 7.6 Hz, 1H), 7.48–7.41 (t, J= 7.5 Hz, 1H), 7.41–7.33 (d, J = 8.2 Hz, 1H), 7.04–6.95 (d, J = 7.5Hz, 2H), 6.78–6.72 (d, J = 8.5 Hz, 1H), 4.90 (s, 1H), 3.69 (s, 3H), 2.90–2.61 (m, 2H), 2.33–2.21 (m, 2H), 2.05 (s, 3H), 1.97–1.80 (m, 2H). 13C NMR (100 MHz, DMSO-d6)δ: 195.44, 160.84, 156.26, 152.46,151.80, 142.18, 138.35, 132.28, 130.21, 126.52, 125.13,124.43, 123.41, 117.30, 113.60, 112.63, 110.36, 102.62, 55.57, 37.20, 33.67, 26.85, 21.25, 16.69. HRMS: m/z calcd for C24H21NO4 [M+Na]+: 410.1363, found: 410.1367.
4v: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), brown solid, mp 208–210 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.85 (s, 1H), 8.38–8.30 (d, J = 7.6 Hz, 1H), 7.72–7.58 (t, J = 7.6 Hz, 1H), 7.50–7.32 (m, 2H), 6.44–6.34 (d, 2H), 6.33–6.24 (d, 1H), 4.97 (s, 1H), 3.66 (s, 6H), 2.95–2.63 (m, 2H), 2.37–2.25 (m, 2H), 2.07–1.75 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.46, 160.90, 160.65, 152.46, 152.36, 148.38, 142.61, 132.44, 124.48, 123.45, 117.33, 113.51, 111.94,106.55, 101.93, 97.76, 55.39, 37.17, 34.37, 26.85, 21.26. HRMS: m/z calcd for C24H21NO5 [M+Na]+: 426.1312, found: 426.1318.
4w: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), brown solid, mp 196–198 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.68 (s, 1H), 8.31 (d, J = 8.0 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.63 (d, J = 8.7 Hz, 1H), 4.99 (s, 1H), 3.72 (s, 3H), 3.69 (s, 3H), 3.65 (s, 3H), 2.73 (m, 2H), 2.24 (m, 2H), 2.06–1.85 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.22, 171.96, 160.55, 152.64, 152.59, 152.47, 151.91,142.69, 142.03, 132.14, 131.35, 126.17, 124.34, 123.24, 117.23, 113.64, 111.94, 107.10, 101.82, 60.68, 60.44, 56.13, 37.23, 32.78, 26.98, 22.97, 21.12. HRMS: m/z calcd for C25H23NO6 [M+Na]+: 456.1418, found: 456.1421. 
4x: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:3, v/v), yellow solid, mp 113–115 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.68 (s, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.99 (s, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 6.75 (s, 2H), 4.84 (s, 1H), 2.85 (d, J = 11.5 Hz, 1H), 2.68 (d, J = 11.2 Hz, 1H), 2.29 (m, 2H), 2.05 (s, 6H), 1.86 (m, 2H). 13C NMR(100 MHz, DMSO-d6) δ: 196.23, 172.83, 162.58, 151.43, 149.58, 143.89, 141.78, 139.46, 135.68, 128.49, 120.38, 117.54, 115.76, 113.51, 111.39, 101.39, 50.60, 34.01, 32.67, 29.50, 27.17, 22.98, 20.85. HRMS: m/z calcd for C25H23NO6 [M+Na]+: 410.1363, found: 410.1368.
4y: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:3, v/v), white solid, mp 320322 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.20 (s, 1H), 8.15 (d, J = 10.8 Hz, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.90 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.37–7.25 (m, 1H), 7.18 (t, J = 8.0 Hz, 1H), 4.90 (s, 1H), 2.28 (m, 2H), 2.20 (m, 2H), 2.02–1.93 (m, 2H),1.75 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ: 196.03,171.96, 163.25, 150.63, 149.38, 142.96, 141.98, 139.87,135.98, 128.68, 120.54, 117.62, 115.79, 113.60, 111.45,101.54, 50.66, 34.25, 32.70, 29.59, 27.18, 23.02, 20.89. HRMS: m/z calcd for C24H21NO4 [M+Na]+: 410.1363, found: 410.1367.
4z: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2,v/v), yellow solid, mp 269–271 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.87 (s, 1H), 8.35–8.25 (d, J = 7.9 Hz, 1H), 7.68–7.68 (t, J = 7.7 Hz, 1H), 7.48–7.34 (m, 2H), 6.52 (s, 2H), 5.00 (s, 1H), 3.68 (s, 6H), 3.58 (s, 3H), 2.95–2.65 (m, 2H), 2.39–2.30 (m, 2H), 2.10–1.90 (m, 10.5 Hz, 2H).13C NMR (100 MHz, DMSO-d6) δ: 195.59, 171.99, 161.02, 152.98, 152.50, 152.43, 142.38,141.72, 136.61, 132.41, 124.47, 123.43, 117.33, 113.54, 111.75, 105.29, 102.16, 60.31, 56.15, 37.24, 34.38, 26.87, 22.96, 21.36. HRMS: m/z calcd for C25H23NO6 [M+Na]+: 456.1418, found: 456.1421.
4aa: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:1, v/v), white solid, mp 296–298 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.70 (s, 1H), 8.30–8.20 (d, J = 7.8 Hz, 1H), 7.68–7.58 (t, J = 7.7 Hz, 1H), 7.44–7.35 (t, J = 7.6 Hz, 2H), 3.96 (d, J = 4.5 Hz, 1H), 2.88–2.52 (m, 2H), 2.45–2.20 (m, 2H), 1.081.85 (m, 2H), 1.68–1.57 (dd, J1 = 11.6 Hz, J2 = 6.7 Hz, 1H), 0.75–0.60 (d, J = 6.8 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ: 196.08, 161.55,153.48, 152.40, 143.91, 132.11, 124.33, 123.04, 117.28, 113.62, 109.99, 100.44, 37.36, 35.08, 33.24, 27.03, 21.13, 19.42, 18.91. HRMS: m/z calcd for C19H19NO3 [M+Na]+: 332.1257, found: 332.1259.
4ab: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:1, v/v), black solid, mp 300–302 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.86 (s, 1H), 8.35–8.27 (d, J = 7.4 Hz, 1H), 7.71–7.60 (t, J = 7.7 Hz, 1H), 7.47–7.38 (m, 3H), 6.30–6.23 (dd, J1 = 3.1Hz, J2 = 1.9 Hz, 1H), 6.04–5.95 (d, J = 3.1 Hz, 1H), 5.16 (s, 1H), 2.88–2.62 (m, 2H), 2.38–2.30 (m, 2H), 2.04–1.91 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.48, 160.76, 156.93, 152.97, 152.44, 143.12, 141.88, 132.63, 124.58, 123.25, 117.41, 113.40,110.90, 109.40, 105.79, 99.12, 37.00, 28.39, 26.81, 21.14. HRMS: m/z calcd for C20H15NO4 [M+Na]+: 356.0893, found: 356.0897. 
4ac: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:1, v/v), yellow solid, mp 351–353 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.92 (s, 1H), 8.34–8.26 (d, J = 7.4 Hz, 1H), 7.68–7.60 (t, J = 8.3 Hz, 1H), 7.47–7.35 (dd,J1 =  14.8 Hz, J2 = 7.8 Hz, 2H), 7.27–7.15 (dd, J1 =  5.1 Hz, J2= 1.1 Hz, 1H), 6.87–6.81 (dd,J1 =  5.0 Hz, J2 = 3.5 Hz, 1H), 6.80–6.72 (d, J = 3.4 Hz, 1H), 5.30 (s, 1H), 2.91–2.64 (m, 2H), 2.40–2.30 (m, 2H), 2.10–1.85 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.57, 160.93, 152.49, 152.42, 149.84, 142.51, 132.65, 127.21, 124.64,124.57, 124.05, 123.34, 117.44, 113.39, 111.81, 101.60, 37.06, 29.27, 26.67, 21.18. HRMS: m/z calcd for C20H15NO3S [M+Na]+: 372.0665, found: 372.0668.
4ad: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:1, v/v), yellow solid, mp 276–278 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.98 (s, 1H), 8.30 (s, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.45 (dd, J1= 13.5Hz, J2 = 7.9 Hz, 2H), 6.93 (d,J = 3.7 Hz, 1H), 6.58 (d, J = 3.6 Hz, 1H), 5.20 (s, 1H), 2.85 (d, J = 7.6 Hz, 1H), 2.69 (d, J = 7.6 Hz, 1H), 2.40–2.34 (m, 2H), 2.05–1.95 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.75, 161.87, 150.57, 147.42, 142.44, 130.47, 132.56, 128.62, 128.35, 128.37, 125.08, 122.60, 116.94, 115.95, 114.87, 103.70, 37.96, 31.27, 29.67, 22.18. HRMS: m/z calcd for C20H14BrNO3S [M+Na]+: 449.9770, found: 449.9772.
4ae: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:1, v/v), white solid, mp 286–288 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.67 (s, 1H), 8.288.20 (d, J = 7.7 Hz, 1H), 7.677.58 (t, J = 7.8 Hz, 1H), 7.45–7.38 (t, J = 7.7 Hz, 2H), 4.00–3.92 (d, J = 4.8 Hz, 1H), 2.89–2.78 (d, J = 7.7 Hz, 1H), 2.64–2.53 (m, 1H), 2.43–2.21 (m, 2H), 2.04–1.89 (m, 2H), 1.64–1.42 (m, J = 14.6 Hz, 4H), 1.34–1.09 (m, 2H), 0.92–0.73 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ: 196.26, 161.61, 153.47, 152.32, 143.89, 132.52, 132.15, 124.40, 124.32, 122.94, 117.29, 113.60, 110.21, 100.44, 45.36, 37.32, 32.79, 29.39, 28.93, 26.98, 26.46, 21.06. HRMS: m/z calcd for C22H23NO3 [M+Na]+: 372.1570, found: 372.1572.
4af: The product was purified with silica gelchromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), yellow solid, mp 342–344 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.77 (s, 1H), 8.32 (d, J = 7.7 Hz, 1H), 7.64 (t, J = 8.1 Hz, 1H), 7.55–7.41 (m, 1H), 7.39 (t, J = 11.6 Hz, 1H), 6.78 (s, 1H), 6.67 (d, J = 4.6 Hz, 2H), 5.88 (s, 2H), 5.02 (s, 1H), 2.902.73 (m, 1H), 2.70–2.60 (m, 1H), 2.26 (t, J = 5.1 Hz, 2H), 2.01–1.84 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 196.90, 161.91, 152.85, 150.42, 147.50, 143.25, 130.28, 127.54, 124.32, 124.01, 122.60, 122.32, 121.84, 119.68, 117.35,115.54, 114.31, 111.25, 101.54, 37.40, 32.68, 29.54, 21.84. HRMS: m/z calcd for C23H17NO5 [M+Na]+: 410.0999, found: 410.1001.
4ag: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), white solid, mp 358–360 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.76 (s, 1H), 8.35–8.29 (d, J = 7.8 Hz, 1H), 7.67–7.61 (t, J = 7.8 Hz, 1H), 7.46–7.42 (t, J = 7.5 Hz, 1H), 7.41–7.36 (d, J = 8.2 Hz, 1H), 6.71–6.64 (s, 3H), 4.88 (s, 1H), 4.15 (s, 4H), 2.92–2.64 (m, 2H), 2.35–2.25 (m, J = 10.3, 2H), 2.03–1.85 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.47, 160.86, 152.45, 151.98, 143.19, 142.34, 142.30, 139.58, 132.37,124.46, 123.43, 120.77, 117.32, 116.99, 116.60, 113.52, 112.34, 102.31, 64.45, 64.37, 37.15, 33.70, 26.81, 21.23. HRMS: m/z calcd for C24H19NO5 [M+Na]+: 424.1155, found: 424.1157.
4ah: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), yellow solid, mp 322–324 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.87 (s, 1H), 8.90–8.75 (d, 1H), 8.45–8.32 (d, 1H), 7.90–7.28 (m, 9H), 5.77 (s, 1H), 2.95–2.70 (m, 2H), 2.32–2.14 (m, 2H), 2.02–1.71 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.91, 161.85, 156.78, 152.46, 152.19, 141.24, 141.01, 136.55, 133.83,129.87, 127.96, 127.47, 127.20, 125.61, 124.64, 123.23, 118.98, 116.84, 114.32, 113.42, 109.58, 102.82,  37.45, 33.47, 29.66, 21.53. HRMS: m/z calcd for C26H19NO3 [M+Na]+: 416.1257, found: 416.1260.
4ai: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:3, v/v), white solid, mp 306–308 °C; 1H NMR (400 MHz, DMSO-d6) δ: 8.22–8.13 (d, J = 8.2 Hz, 1H), 7.90–7.76 (dd, J1 = 10.2 Hz, J2 = 8.8 Hz, 4H), 7.54–7.45 (t,J = 7.6 Hz, 1H), 7.45–7.21 (m, 4H), 7.16–7.08 (d,J = 8.1 Hz, 1H), 5.57 (s, 1H), 2.79–2.63 (m, 2H), 2.42–2.29 (m, 2H), 2.01 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 196.85, 161.19, 156.80, 152.78, 153.69, 141.84, 141.71, 138.95, 135.84, 128.97, 127.69, 127.84, 127.68, 125.91, 123.98, 123.86, 119.12, 117.68, 115.21, 114.32, 108.85, 102.54, 37.68, 33.54, 29.80, 21.42. HRMS: m/z calcd for C26H19NO4 [M+Na]+: 432.1206, found: 432.1212.
4aj: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), white solid, mp 368–370 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.84 (s, 1H), 8.42–8.33 (d, J = 7.9 Hz, 1H), 7.77–7.73 (d, J = 9.0 Hz, 1H), 7.70–7.55 (dd, J1= 11.8Hz, J2 = 7.9 Hz, 3H), 7.50–7.35 (m, J = 10.2 Hz,3H), 7.24–7.18 (d, J = 2.2 Hz, 1H), 7.12–7.03 (dd, J1 = 9.0 Hz, J2 = 2.4 Hz, 1H), 5.13 (s, 1H), 3.82 (s, 3H), 2.93–2.68 (m, 2H), 2.35–2.21 (m, 2H), 2.03–1.85 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 195.51, 160.90, 157.38, 152.56, 152.25, 142.64, 141.64, 133.50, 132.38, 129.73, 128.63, 127.55, 127.02, 126.15, 124.46, 123.53, 118.84, 117.34, 113.63, 112.34, 105.98, 102.22, 55.58, 37.27, 34.74, 26.96, 21.23. HRMS: m/z calcd for C27H21NO4 [M+Na]+: 446.1363, found: 446.1366.
4ak: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), white solid, mp 336–338 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.72 (s, 1H), 8.32 (d, J = 7.8 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 7.22 (dt, J1 = 8.0 Hz, J2 = 7.4 Hz, 4H), 7.10 (t, J = 7.0 Hz, 1H), 4.95 (s, 1H), 2.67 (s, 2H), 2.28 (m, 1H), 2.09 (m, 1H), 1.08 (s, 3H), 0.94 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 195.14, 171.95, 160.73, 152.50, 150.14, 146.27, 142.57, 132.41, 128.43, 128.22, 126.70, 124.50, 123.41, 117.32, 113.49, 111.29, 102.31, 50.57, 34.92, 32.65, 29.55, 27.00, 22.99. HRMS: m/z calcd for C24H21NO3 [M+Na]+: 394.1414, found: 394.1416.
4al: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), yellow solid, mp 216–218 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.67 (s, 1H), 8.30 (d, J = 7.9 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 4.89 (s, 1H), 3.66 (s, 3H), 2.65 (s, 2H), 2.27 (m, 1H), 2.08 (m, 1H), 1.07 (s, 3H), 0.95 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 195.25, 160.77,158.13, 152.45, 149.90, 142.30, 138.60, 132.35, 129.18, 124.51, 123.34, 117.31, 113.79, 113.51, 111.47, 102.60, 55.37, 50.58, 33.97, 32.64, 29.56, 27.02. HRMS: m/z calcdfor C25H23NO4 [M+Na]+: 424.1519, found: 424.1522.
4am: The product was purified with silica gel chromatography (Petroleum ether–Ethyl acetate, 1:2, v/v), yellow solid, mp 212–214 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.67 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 6.68 (s, 3H), 4.84 (s, 1H), 4.15 (s, 4H), 2.66 (d, J = 4.8 Hz, 2H), 2.51 (s, 1H), 2.27 (m, 1H), 2.11 (m, 1H), 1.77 (s, 1H), 1.07 (s, 3H), 0.98 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 195.17, 171.93, 160.76, 152.45, 149.99, 143.14, 142.31, 142.28, 139.46, 132.35, 124.49, 123.35, 120.83, 117.31, 116.95, 116.73, 113.51, 111.23, 102.39, 64.44, 50.60, 34.01, 32.67, 29.50, 27.17, 22.98. HRMS: m/z calcd for C26H23NO5 [M+Na]+: 452.1468, found: 452.1473.
4.3. General procedure for the anti-AChE activity assays
The inhibitory ability of chromeno[4,3-b]quinoline derivatives against acetylcholinesterase (AChE, E.C. 3.1.1.7, from the electric eel) was tested using the 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) method. DTNB would generate 5-mercapto-2-nitrobenzoic acid-a detectable chromophore at the 405–412 nm range. To evaluate the biological activity, the tested compounds were dissolved in DMSO to an initial concentration of 101 mol/L, and then they were diluted into six different concentrations by a mixture of DMSO and EtOH (DMSOEtOH, 1:9, v/v), which could be tested toobtain the range of 20% to 80% enzyme inhibition for AChE. For this purpose, in a 96-well plate, 26 μL phosphate buffer (0.1 M, pH = 8.0), 30 μL of DTNB 0.01 M, 4 μL of enzyme [5 U/mL of AChE] and 10 μL of inhibitor solution or DMSOEtOH mixture (1:9, v/v) were added. Then, 30 μL of 0.01 M substrate (acetylthiocholine iodide) was added to each well, and the change of absorbance was measured at 410 nm for 2 min. Each experiment was done in triplicate. The IC50 values were graphically determined from inhibition curves (log inhibitor concentration vs. percent of inhibition).
4.4. General procedure for the molecular modelling
Because of the lack of the complex crystal structure of the compound, compound 4ag and Donepezil with the acetylcholinesterase-related protein (PDB: 1W75) were used as a model in the docking experiments. Autodock Vina was chosen to study the binding modes of the compound 4ag, Donepezil and their differences. Heteroatoms and water molecules in the proteins were removed, and hydrogen atoms were subsequently added.
Supporting Information Available: Copies of 1H NMR, 13C NMR spectral and X-ray data.
Acknowledgements
We are grateful to the NSFC (Grant No. 81773557, 81573279, 81373255), Major Project of Technology Innovation Program of Hubei Province (Grant No. 2016ACA126), NSFHP (Grant No. 2017CFA024), and the Fundamental Research Funds for the Central Universities of China (Grant No. 2042017kf0288) for support of this research.
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喹啉-苯并吡喃类新型乙酰胆碱酯酶抑制剂的微波一锅法的设计合成以及生物活性研究
贺明1#, 谢宝花1#, 贺培1, 周海兵1*, 黄胜堂2*, 董春娥1*
1. 武汉大学 药学院,湖北 武汉 430072
2. 湖北科技学院药学院, 湖北咸宁437100
摘要: 在微波条件下,4-羟基香豆素、甲醛、环己二酮、硝酸铈胺为原料一锅法高产率的合成系列喹啉-苯并吡喃类衍生物。化合物的结构通过X-ray单晶衍射确定。通过经典的Ellman,分别测试了20 μM50 μM浓度条件下所合成化合物对乙酰胆碱酯酶的抑制活性,并系统总结了该类化合物详细的构效关系。其中化合物4ag呈现出很好的抑制活性,IC50值达到5.63 µM此外,通过对化合物与乙酰胆碱酯酶的分子模拟研究,初步探究了该类化合物可能的作用机理。 
关键词: 苯并吡喃-喹啉类化合物; 抗乙酰胆碱酯酶活性; 分子模拟; 四组分反应
 
 
 
 
Received: 2018-03-18, Revised: 2018-05-29, Accepted: 2018-09-17.
Foundation items: NSFC (Grant No. 81773557, 81573279, 81373255), Major Project of Technology Innovation Program of Hubei Province (Grant No. 2016ACA126), NSFHP (Grant No. 2017CFA024), and the Fundamental Research Funds for the Central Universities of China (Grant No. 2042017kf0288).
#The two authors contributed equally.
*Corresponding author. Tel.: +86-027-68759586, E-mail: zhouhb@whue.du.cn; cdong@whu.edu.cnhuangst6511@hbust.edu.cn         
 
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