Synthesis and Evolution of Berberine Derivatives as a New ... - MDPI

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Synthesis and Evolution of Berberine Derivatives as a New Class of Antiviral Agents against Enterovirus 71 through the MEK/ERK Pathway and Autophagy Yan-Xiang Wang † ID , Lu Yang † , Hui-Qiang Wang, Xiao-Qiang Zhao, Ting Liu, Ying-Hong Li, Qing-Xuan Zeng, Yu-Huan Li * and Dan-Qing Song * Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China; [email protected] (Y.-X.W.); [email protected] (L.Y.); [email protected] (H.-Q.W.); [email protected] (X.-Q.Z.); [email protected] (T.L.); [email protected] (Y.-H.L.); [email protected] (Q.-X.Z.) * Correspondence: [email protected] (Y.-H.L.); [email protected] (D.-Q.S.); Tel.: +86-10-6301-0984 (Y.-H.L.); +86-10-6316-5268 (D.-Q.S.) † These authors contributed equally to this work. Received: 17 July 2018; Accepted: 16 August 2018; Published: 20 August 2018







Abstract: Taking berberine (BBR) as the lead, 23 new BBR derivatives were synthesized and examined for their antiviral activities against four different genotype enterovirus 71 (EV71) strains with a cytopathic effect (CPE) assay. Structure-activity relationship (SAR) studies indicated that introduction of a suitable substituent at the 9-position might be beneficial for potency. Among them, compound 2d exhibited most potent activities with IC50 values of 7.12–14.8 µM, similar to that of BBR. The effect of 2d was further confirmed in a dose-dependent manner both in RNA and protein level. The mechanism revealed that 2d could inhibit the activation of MEK/ERK signaling pathway. Meanwhile, it could suppress the EV71-induced autophagy by activating AKT and inhibiting the phosphorylation of JNK and PI3KIII proteins. We consider BBR derivatives to be a new family of anti-EV71 agents through targeting host components, with an advantage of broad-spectrum anti-EV71 potency. Keywords: enterovirus 71; berberine; structure-activity relationship; MEK/ERK pathway; autophagy

1. Introduction Enterovirus 71 (EV71), a single-stranded positive-sense RNA virus belonging to the enterovirus genus of the Picornaviridae family, is the primary cause of the hand, foot and mouth disease (HFMD), which is widely spread among infants and young children, especially those under 5 years old [1,2]. EV71 was first isolated from patients with central nervous system (CNS) diseases in California in 1969 and HFMD caused by EV71 infections usually is self-limiting, but some EV71-caused HFMD cases have been associated with neurological diseases such as aseptic meningitis, myocarditis and pulmonary edema [3–5]. Every year, from April to July, HFMD has a high incidence, which has become a seriously social and public health concern in mainland China. However, up to now there is still no effective drug for the prevention and treatment of HFMD in the clinic. This situation has resulted in a very pressing need for the discovery of novel anti-EV71 drug candidates for the control of infectious diseases arising from EV71 [6,7]. We have been working to find innovative drug candidates from Chinese natural products such as berberine (BBR, Figure 1), which has been widely used in China for decades against diarrhea as a Traditional Chinese Medicine (TCM). We have continuously reported that BBR derivatives possess various pharmacological effects, such as antibacterial [8], anti-Coxsackie virus [9], Molecules 2018, 23, 2084; doi:10.3390/molecules23082084

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Traditional Chinese Medicine (TCM). We have continuously reported that BBR derivatives possess Molecules 2018, 23, x 16 various pharmacological effects, such as antibacterial [8], anti-Coxsackie virus 2 of [9], anti-inflammatory [10], immunotherapeutic [11] and anti-tuberculosis activities [12]. Recently, we Traditional Chinese Medicine (TCM). We have continuously reported that BBR derivatives possess Molecules 2018,identified 23, 2084 of 16 have first that BBR exerted a moderate activity against EV71 replication with 2new various pharmacological effects, such as antibacterial [8], anti-Coxsackie virus [9], mechanism of action, mainly through down-regulating EV71-induced autophagy and MEK/ERK anti-inflammatory [10], immunotherapeutic [11] and anti-tuberculosis activities [12]. Recently, we signaling pathway [4]. The unique chemical scaffold and biological activity against EV71 of BBR anti-inflammatory [10],that immunotherapeutic and anti-tuberculosis activities Recently, we have have first identified BBR exerted a [11] moderate activity against EV71 [12]. replication with new spurred us to further conduct structural modifications and optimization of its kind, so as to explore first identified BBRmainly exertedthrough a moderate activity against EV71 replication with new mechanism of that action, down-regulating EV71-induced autophagy andmechanism MEK/ERK structure-activity relationship (SAR) against EV71 as well as acquire promising anti-EV71 of action, pathway mainly through autophagy and MEK/ERK signaling signaling [4]. The down-regulating unique chemical EV71-induced scaffold and biological activity against EV71 of BBR candidates. pathway [4].toThe unique chemical scaffold and biological activity against of EV71 of BBR us to spurred us further conduct structural modifications and optimization its kind, sospurred as to explore further conduct structural modifications and optimization of its kind, so as to explore structure-activity structure-activity relationship (SAR) against EV71 as well as acquire promising anti-EV71 relationship candidates. (SAR) against EV71 as well as acquire promising anti-EV71 candidates.

Figure 1. Chemical structure of BBR, and structure modification strategy.

Based on the strategy, a series of new BBR derivatives, including variety esters and ethers on 1. Chemical structure of BBR, BBR, and structure structure modification strategy. Figure 1. Chemical structure of and positions 3 and 9 Figure as depicted in Figure 1, were prepared due to modification the startingstrategy. material availability and synthetic ease, and evaluated for their anti-EV71 activity taking BBR as the lead. In the present Based on the strategy, a series of new BBR derivatives, including variety esters and ethers on study, we described the synthesis 23new newBBR BBRderivatives, derivatives, including SAR analysis and esters primary mechanism Based on the strategy, a seriesofof variety and ethers on positions 3 and 9 as depicted in Figure 1, were prepared due to the starting material availability and of action of the representative compound. positions 3 and 9 as depicted in Figure 1, were prepared due to the starting material availability and synthetic ease, and evaluated for their anti-EV71 activity taking BBR as the lead. In the present synthetic ease, and evaluated for their anti-EV71 activity taking BBR as the lead. In the present study, study, we and described the synthesis of 23 new BBR derivatives, SAR analysis and primary mechanism 2. Results Discussion we described the synthesis of 23 new BBR derivatives, SAR analysis and primary mechanism of action of action of the representative compound. of the representative compound. 2.1. Chemistry 2. Results and Discussion 2. Results and A total of Discussion twenty-three new BBR derivatives were semi-synthesized as displayed in Schemes 1–3, taking commercially available BBR, palmatine (PMT) or jatrorrhizine (JTH) as the starting 2.1. Chemistry Chemistry 2.1. material, respectively. All the 1H-NMR, 13C-NMR, HRMS-ESI spectra data of target compounds can A total totalof oftwenty-three twenty-threenew newBBR BBR derivatives were semi-synthesized as displayed in Schemes A derivatives semi-synthesized as displayed Schemes 1–3, be found in Supplementary Materials. As shownwere in Scheme 1, the key intermediate 1inwas obtained 1–3, taking commercially available BBR, palmatine (PMT) or jatrorrhizine (JTH) as the starting taking commercially BBR, palmatine (PMT) or jatrorrhizine (JTH) as thewere starting material, under vacuum from available BBR as reported previously [10,11]. Then, BBR ethers 2a–g obtained by 13C-NMR, HRMS-ESI spectra data of target compounds can 1 H-NMR, material, respectively. All the 113 H-NMR, respectively. All the C-NMR, HRMS-ESI spectra data of target compounds can be found alkylation of compound 1 with corresponding bromides with K2CO3 as the base with yields of be Supplementary found in Supplementary Materials. Asinshown in Scheme 1, the key intermediate 1 was obtained in Scheme 1, the key intermediate 1 was obtained under 40–58%. Similarly, Materials. BBR esters As 2h shown and 2i were prepared through esterification of compound 1 [13] under vacuum from BBR as reported previously [10,11]. Then, BBR ethers 2a–g were obtained by vacuum from BBR aschlorides reported in previously Then, BBR ethers 2a–g were obtained by alkylation with corresponding 37% and [10,11]. 38% yield, respectively. alkylation of compound 1 with corresponding bromides with K 2 CO 3 as the base with yields of of compound 1 with bromides with as the base with of 40–58%. 2 CO3synthesized As described incorresponding Scheme 2, intermediate 3 [14]Kwas after yields demethylation ofSimilarly, PMT on 40–58%. Similarly, BBR esters 2h and 2i wereesterification prepared through esterification of compound 1 [13] BBR esters 2h and 2i were prepared through compound [13]base with position 9. Compounds 4a–f were prepared via alkylation of with K2CO3 as1the incorresponding overall yields with corresponding chlorides in 37% and 38% yield, respectively. chlorides 37% and 38% yield, respectively. of 20–29%,inwhile compound 4g was created by esterification of compound 3 by 21% yield. As described in Scheme 2, intermediate 3 [14] was synthesized after demethylation of PMT on position 9. Compounds 4a–f were prepared via alkylation with K2CO3 as the base in overall yields of 20–29%, while compound 4g was created by esterification of compound 3 by 21% yield.

Scheme 1. 1. Reagents Reagentsand andconditions: conditions:(a)(a) 195 30–40 mmHg, h; R(b)Br,RK 1Br, K2CO3, DMF, ◦ C,°C, ◦ 70 °C; (c) Scheme 195 30–40 mmHg, 1 h;1(b) 1 2 CO3 , DMF, 70 C; (c) R2 Cl, ◦ R 2Cl, K2CO3, CH3CN, 70 °C. K2 CO3 , CH3 CN, 70 C. Scheme 1. Reagents and conditions: (a) 195 °C, 30–40 mmHg, 1 h; (b) R1Br, K2CO3, DMF, 70 °C; (c) R2Cl, K2CO3, CH3CN, 70 °C.

Molecules 2018, 23, 2084 Molecules 2018, 23, x Molecules 2018, 23, x

3 of 16 3 of 16 3 of 16

Scheme 2. Reagents Reagents andconditions: conditions: (a) 195 30–40 mmHg, 1 h; (b) R Br, K,2DMF, CO3, DMF, 70 °C; (c) ◦ C,°C, Scheme (a)(a) 195195 30–40 mmHg, 1 h; (b) Br, R K112Br, COK 70 ◦ C; (c) 3 2CO3, DMF, Scheme 2. 2. Reagentsand and conditions: °C, 30–40 mmHg, 1 h;R1(b) 70 Benzyl °C; (c) Benzyl chloroformate, K 2CO3, CH3CN, 70 °C. ◦ chloroformate, K CO , CH CN, 70 C. 2 3 3 Benzyl chloroformate, K2CO3, CH3CN, 70 °C.

Scheme 3. Reagents and conditions: (a) R1Br, K2CO3, DMF, room temperature; (b) Benzyl Scheme 3.3.Reagents Reagents and conditions: (a) KRCO 1Br, K2CO3, DMF, room temperature; (b) Benzyl Scheme and conditions: (a) R1 Br, 2 3 , DMF, room temperature; (b) Benzyl chloroformate, chloroformate, K2CO 3, CH3CN, room temperature. chloroformate, K 2CO3, CH3CN, room temperature. K2 CO3 , CH3 CN, room temperature.

Finally, as depicted in Scheme 3, the ether products 5a–f were directly obtained via alkylation Finally, as depicted in Scheme 3, the ether products 5a–f were directly obtained via alkylation usingAs corresponding bromides room temperature from starting material JTH. JTH wasofesterified described in Scheme 2, at intermediate 3 [14] was synthesized after demethylation PMT on using corresponding bromides at room temperature from starting material JTH. JTH was esterified with benzyl chloroformate alkaline condition to obtain by in 36% yield.yields All the position 9. Compounds 4a–funder were prepared via alkylation with Kcompound base overall of 2 CO3 as the5g with benzyl chloroformate under alkaline condition to obtain compound 5g by 36% yield. All the final products were purified flash by column chromatography using 20–29%, while compound 4g wasvia created esterification of compound 3 by CH 21%3OH/CH yield. 2Cl2 as the final products were purified via flash column chromatography using CH3OH/CH2Cl2 as the gradient eluent. Finally, as depicted in Scheme 3, the ether products 5a–f were directly obtained via alkylation gradient eluent. using corresponding bromides at room temperature from starting material JTH. JTH was esterified 2.2. Pharmacological Evaluation with benzyl chloroformate under alkaline condition to obtain compound 5g by 36% yield. All the 2.2. Pharmacological Evaluation final products were purified via flash column chromatography using CH3 OH/CH2 Cl2 as the 2.2.1. SAR for Anti-EV71 Activity gradient 2.2.1. SAReluent. for Anti-EV71 Activity Thus, all newly synthesized BBR analogues were screened for their anti-EV71 potencies in Thus, all newly synthesized BBR analogues were screened for their anti-EV71 potencies in 2.2. Pharmacological Evaluation African green monkey kidney (Vero) cells taking BBR as the positive control. EV71 strains used in African green monkey kidney (Vero) cells taking BBR as the positive control. EV71 strains used in the present study include different genotype strains, such as H strain (VR-1432, genotype C2), BrCr 2.2.1. SAR for Anti-EV71 the present study includeActivity different genotype strains, such as H strain (VR-1432, genotype C2), BrCr strain (VR-1775, genotype A), and JS-52 and SHZH98 (genotype C4). The potency against EV71 strainThus, (VR-1775, genotype A), and JS-52 and SHZH98 (genotype C4). The potency against EV71 all newly synthesized analogues werewas screened for their anti-EV71 potencies African strains above mentioned of eachBBR tested compound evaluated by the combination of itsinIC 50 and strains above mentioned of each tested compound was evaluated by the combination of its IC 50 and green monkey kidney (Vero) as cells taking BBR astherapeutic the positiveindication. control. EV71 usedand in the present selectivity index (SI) value the important Thestrains structure anti-EV71 selectivity index (SI) value as the important therapeutic indication. The structure and anti-EV71 study include different genotype strains, such H strain activity of each new compound are shown inas Table 1. (VR-1432, genotype C2), BrCr strain (VR-1775, activity of each new compound are shown in Table 1. genotype and SHZH98 C4). Thescreened potency against strains First,A), theand keyJS-52 intermediates 1, (genotype 3, and JTH were againstEV71 H strain of above EV71, mentioned and all of First, the compound key intermediates 1, 3, and JTHcombination were screened against H strain of EV71, and all of of each tested was evaluated by the of its IC and selectivity (SI)on value 50 them showed decreased activity compared with that of BBR. SAR analysis was thenindex focused the them decreased activity compared with that of BBR. SAR analysis thennew focused on the as theshowed important therapeutic structure anti-EV71 activitywas of each influence of substitutions onindication. position 9 The of ring D, andand seven ether derivatives (2a–g) andcompound two ester influence substitutions on position 9 of ring D, and seven ether derivatives (2a–g) and two ester are shownofin analogues (2hTable and1.2i) were prepared and tested, respectively. As described in the table, among analogues (2h and 2i) were prepared and tested, respectively. As described in the table, among them, ether compounds 2d–g with different substituents on the benzene ring displayed satisfactory them, ether compounds 2d–g with different substituents on the benzene ring displayed satisfactory potencies with IC50 values of 12.4 and 15.8 μM, especially compound 2d that exhibited lower potencies with IC50 values of 12.4 and 15.8 μM, especially compound 2d that exhibited lower cytotoxicity and a better SI value of 6.7, similar to that of BBR. Meanwhile, the carboxylic acid form cytotoxicity and a better SI value of 6.7, similar to that of BBR. Meanwhile, the carboxylic acid form of compound 2d (2d-a) exhibited a complete loss on the antivirus potency which indicated that of compound 2d (2d-a) exhibited a complete loss on the antivirus potency which indicated that compound 2d might exert the activity on its original form. Two ester derivatives 2h and 2i gave compound 2d might exert the activity on its original form. Two ester derivatives 2h and 2i gave obvious decreased activity, The results hinted that introducing a suitable ether substituent on obvious decreased activity, The results hinted that introducing a suitable ether substituent on position 9 might be beneficial for the ability against EV71. position 9 might be beneficial for the ability against EV71.

Molecules 2018, 23, Molecules 2018, 2018, 23, 23, x x Molecules 2018, 23, xx Molecules Molecules Molecules 2018, 23, xx Molecules 2018, 2018, 23, 23, x

4 of 16 4 of of 16 16 of 16 4444 of 16 4 of of 16 16

Molecules 2018, 23, xx 44 of 16 Molecules 2018, 23, of 16 Molecules 2018, 23, 2084 4 of 16 Molecules 2018, 23, xx 44 of of 16 Molecules 2018, 23, x of 16 16 Molecules 2018, 23, Molecules 2018, 23, x 444 of 16 Table 1. SAR of all target compounds for anti-EV71 activity and cytotoxicity in Vero cells. Molecules 2018, 23, x of 16 Molecules 2018, 23, x 4 of 16 Molecules 2018, 23, x 4 of 16 Table 1. SAR of all target compounds for anti-EV71 activity and cytotoxicity in Vero cells. Molecules 2018, 23, x1. 4 of 16 Table SAR of all target compounds for anti-EV71 activity and cytotoxicity in Vero cells. Table 1. SAR of all target compounds for anti-EV71 activity and cytotoxicity in Vero cells. Table 1. SAR of all target compounds for anti-EV71 activity and cytotoxicity in Vero cells. Table 1. SAR of all target compounds for anti-EV71 activity and cytotoxicity in Vero cells. Table 1. SAR of all target compounds for anti-EV71 activity and cytotoxicity in Vero cells. Table 1. SAR of all target compounds for anti-EV71 activity and cytotoxicity in Vero cells. Table 1. 1. SAR SAR of of all all target target compounds compounds for for anti-EV71 anti-EV71 activity activity and and cytotoxicity cytotoxicity in in Vero Vero cells. cells. Table Table SAR of all target compounds for anti-EV71 activity and cytotoxicity in Vero cells. Table 1. SAR of all target compounds for anti-EV71 activity and cytotoxicity in Vero cells. Table 1. SAR of all target compounds activity and cytotoxicity in Vero cells. Table 1.1. of compounds for anti-EV71 cytotoxicity in cells. Table 1.SAR SAR ofall alltarget target compoundsfor foranti-EV71 anti-EV71activity activityand and cytotoxicity inVero Vero cells.

No. R No. R No. R No. R No. R No. R No. R No. RBBR No. R No. R BBR No. BBR ---BBR No. No. RR BBR R 1No. No. BBR --BBR 1 BBR 1 BBR 1113 BBR ---BBR 1 BBR 3 BBR 1 BBR 13133133 ---JTH BBR 1 JTH 1 3 JTH JTH JTH 3331 1 --JTH JTH 33 32a ----JTH JTH 3 JTH 2a JTH 2a JTH 2a JTH ----JTH 2a 2a JTH 2a 2a 2a 2a 2a 2a 2a 2b 2a2a 2b 2b 2b 2b 2b 2b 2b 2b 2b 2b 2b 2b 2b 2b 2c 2c 2c 2c 2c 2c 2c 2c 2c 2c 2c 2c 2c 2c 2c 2d 2d 2d 2d 2d 2d 2d 2d 2d 2d 2d 2d 2d-a 2d 2d 2d 2d-a 2d-a 2d-a 2d-a 2d-a 2d-a 2d-a 2d-a 2d-a 2d-a 2d-a 2e 2d-a 2d-a 2d-a 2e 2e 2e 2e 2e 2e 2e2e 2e 2e 2e 2e 2e 2e 2f 2f 2f 2f 2f 2f 2f 2f2f 2f 2f 2f 2f 2f 2f 2g 2g 2g 2g 2g 2g 2g 2g 2g 2g 2g 2g 2g 2g 2g 2h 2h 2h 2h 2h 2h 2h 2h 2h 2h 2h 2h 2i 2h 2h 2h 2i 2i 2i 2i 2i 2i 2i 2i 2i 2i 2i 4a 2i 2i 2i 4a 4a 4a 4a 4a 4a 4a 4a 4a 4a 4a 4a 4a 4a 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4b 4c 4c4c 4c 4c 4c 4c 4c 4c 4c 4c 4c 4c 4c 4c 4d 4d 4d 4d 4d 4d 4d 4d 4d 4d 4d 4d 4d 4d 4d 4e 4e4e 4e 4e 4e 4e 4e 4e 4e 4e 4e 4e 4e 4e 2018, 23, x Molecules 4f 4f4f 4f 4f 4f 4f 4f 4f 4f 4f 4f 4f 4f 4g 4f4g

a 50 TC50 50 aa TC 50 aaa 50 TC a 50 TC a 50 TC a 50 TC a 50 TC 50 TC 147 50 aaa 50 TC 50 TC 147 50 TC 147 aaaaa 147 50 TC 50 TC TC 147 85.5 50 50 TC 147 147 85.5 147 85.5 147 85.5 85.5 147 254 85.5 147 85.5 147 254 147 85.5 147 254 85.5 254 254 85.5 255 147 254 85.5 254 85.5 255 85.5 254 85.5 255 254 255 255 254 85.5 255 254 255 254 254 255 254 255 223 254 255 223 255 223 255 223 255 255 223 223 255 223 223 223 223 223 223 223 22.3 223 223 22.3 22.3 22.3 22.3 22.3 22.3 22.3 22.3 22.3 22.3 22.3 22.3 22.3 22.3 27.9 27.9 27.9 27.9 27.9 27.9 27.9 27.9 27.9 27.9 27.9 27.9 27.9 27.9 27.9 82.8 82.8 82.8 82.8 82.8 82.8 82.8 82.8 82.8 82.8 82.8 82.8 437 82.8 82.8 82.8 437 437 437 437 437 437 437 437 437 437 437 14.4 437 437 437 14.4 14.4 14.4 14.4 14.4 14.4 14.4 14.4 14.4 14.4 14.4 14.4 14.4 14.4 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 9.22 54.9 54.9 54.9 54.9 54.9 54.9 54.9 54.9 54.9 54.9 54.9 54.9 54.9 54.9 54.9 124 124 124 124 124 124 124 124 124 124 124 124 167 124 124 124 167 167 167 167 167 167 167 167 167 167 167 434 167 167 167 434 434 434 434 434 434 434 434 434 434 434 434 434 434 333 333 333 333 333 333 333 333 333 333 333 333 333 333 333 452 452 452 452 452 452 452 452 452 452 452 452 452 452 452 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 143 143 143 143 143 143 143 143 143 143 143 143 143 143 143 306 306 306 306 306 306 306 306 306 306 306 306 306 306 351 351 306

H Strain H Strain Strain H H aStrain H Strain 50 SI bb H IC 50 aaaStrain H Strain 50 a H Strain IC 50 SI bbbbb a IC 50 SI H Strain aStrain IC 50 SI b a H IC 50 SI b aaStrain IC 50 SI H 21.2 6.93 IC 50 SI H aStrain H IC 50 SI H Strain 21.2 6.93 H IC 50Strain SI bbbbb 21.2 6.93 aaaStrain 50 21.2 6.93 IC 50 SI 21.2 6.93 37.0 2.31 21.2 6.93 IC 50a a SI b b 21.2 6.93 37.0 2.31 a IC 50 SI IC 5050 SI 21.2 6.93 a IC SIbb 37.0 2.31 21.2 6.93 IC 37.0 2.31 SI 50 37.0 2.31 21.2 6.93 121 2.10 37.0 2.31 21.2 6.93 37.0 2.31 21.2 6.93 121 2.10 21.2 6.93 37.0 2.31 21.2 6.93 121 2.10 37.0 2.31 121 2.10 121 2.10 37.0 2.31 >147 21.2 6.93 121 2.10 37.0 2.31 121 2.10 37.0 2.31 >147 37.0 2.31 121 2.10 37.0 2.31 >147 ---121 2.10 >147 >147 121 2.10 37.0 2.31 >147 121 2.10 >147 121 2.10 121 2.10 >147 121 2.10 >147 48.3 4.62 121 2.10 >147 --48.3 4.62 >147 48.3 4.62 >147 48.3 4.62 >147 >147 48.3 4.62 48.3 4.62 >147 -48.3 4.62 48.3 4.62 48.3 4.62 48.3 4.62 48.3 4.62 48.3 4.62 48.3 4.62 >5.34 48.3 4.62 48.3 4.62 >5.34 >5.34 --->5.34 >5.34 >5.34 -->5.34 >5.34 >5.34 >5.34 ->5.34 >5.34 >5.34 >5.34 ---->5.34 13.8 2.02 13.8 2.02 13.8 2.02 13.8 2.02 13.8 2.02 13.8 2.02 13.8 2.02 13.8 2.02 13.8 2.02 13.8 2.02 13.8 13.8 2.02 13.8 2.02 13.8 2.02 13.8 2.02 12.4 6.70 12.4 6.70 12.4 6.70 12.4 6.70 12.4 6.70 12.4 6.70 12.4 6.70 12.4 6.70 12.4 6.70 12.4 12.4 6.70 12.4 6.70 >145 12.4 6.70 12.4 6.70 12.4 6.70 >145 >145 --->145 >145 >145 >145 >145 >145 >145 --->145 >145 >5.31 >145 >145 >145 >5.31 >5.31 -->5.31 >5.31 >5.31 -->5.31 >5.31 >5.31 -->5.31 >5.31 >5.31 >5.31 >5.31 --->5.31 >5.33 >5.33 >5.33 --->5.33 >5.33 >5.33 >5.33 >5.33 >5.33 --->5.33 >5.33 >5.33 >5.33 >5.33 --->5.33 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 15.8 3.47 >47.9 >47.9 >47.9 --->47.9 >47.9 >47.9 >47.9 >47.9 ->47.9 >47.9 >47.9 ->47.9 31.1 5.37 >47.9 >47.9 --->47.9 31.1 5.37 31.1 5.37 31.1 5.37 31.1 5.37 31.1 5.37 31.1 5.37 31.1 5.37 31.1 5.37 31.1 5.37 31.1 5.37 31.1 5.37 31.1 5.37 31.1 5.37 144 3.01 31.1 5.37 144 3.01 144 3.01 144 144 3.01 144 3.01 144 3.01 144 3.01 144 3.01 144 3.01 144 3.01 144 3.01 144 3.01 144 3.01 144 3.01 >144 >144 >144 >144 -->144 >144 >144 >144 ->144 >144 >144 ->144 >144 >144 --->144 >143 >143 >143 -->143 >143 >143 ->143 >143 >143 >143 >143 ->143 >143 >143 --->143 >15.9 >15.9 -->15.9 >15.9 >15.9 -->15.9 >15.9 >15.9 ->15.9 >15.9 >15.9 ->15.9 >15.9 >15.9 --->15.9 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 13.6 10.5 82.2 3.73 82.2 3.73 82.2 3.73 82.2 3.73 82.2 3.73 82.2 3.73 82.2 3.73 82.2 3.73 82.2 3.73 82.2 3.73 82.2 3.73 82.2 3.73 82.2 3.73 82.2 3.73 >143 >143 82.2 3.73

JS-52 Strain SHZH98 Strain JS-52 Strain Strain SHZH98 Strain Strain JS-52 SHZH98 JS-52 SHZH98 Strain aa Strain a JS-52 Strain SHZH98 Strain 50 SI IC50 50 SI JS-52 Strain SHZH98 Strain a IC 50 50 aaa JS-52 Strain SHZH98 Strain 50 aa a JS-52 Strain SHZH98 Strain IC 50 SI IC 50 SI a a IC 50 SI IC 50 SI JS-52 Strain SHZH98 Strain IC 50 SI IC 50 SI JS-52 Strain SHZH98 Strain IC 50 aaa SI IC 50 aaa SI 10.5 28.2 5.22 IC 50 SI IC 50 SI JS-52 Strain SHZH98 Strain 14.0 a a IC 50 SI IC 50 SI JS-52 Strain SHZH98 Strain a Strain JS-52 SHZH98 Strain IC 50 SI IC 50 SI JS-52 Strain SHZH98 Strain 14.0 10.5 28.2 5.22 JS-52 Strain SHZH98 Strain IC 50 SI IC 50 aaa SI 14.0 10.5 28.2 5.22 aa 50 50 14.0 10.5 28.2 5.22 IC 50 SI IC 50 SI 14.0 10.5 28.2 5.22 28.5 3.00 32.6 2.62 14.0 10.5 28.2 5.22 IC 50aaa SI IC 50aaa SI 14.0 10.5 28.2 5.22 28.5 3.00 32.6 2.62 a a a a IC SI IC SI IC 5050 SI IC 5050 SI 14.0 10.5 28.2 5.22 IC 50 SI IC 50 SI 28.5 3.00 32.6 2.62 14.0 10.5 28.2 5.22 IC50 IC50 SI 28.5 3.00 32.6 2.62 28.5 3.00 32.6 2.62 14.0 10.5 28.2 5.22 102 2.50SI 132 1.93 28.5 3.00 32.6 2.62 14.0 10.5 28.2 5.22 28.5 3.00 32.6 2.62 14.0 10.5 28.2 5.22 102 2.50 132 1.93 14.0 10.5 28.2 5.22 28.5 3.00 32.6 2.62 14.0 10.5 28.2 5.22 102 2.50 132 1.93 28.5 3.00 32.6 2.62 102 2.50 132 1.93 102 2.50 132 1.93 28.5 3.00 32.6 2.62 147 1.73 147 1.73 14.0 10.5 28.2 5.22 102 2.50 132 1.93 28.5 3.00 32.6 2.62 102 2.50 132 1.93 28.5 3.00 32.6 2.62 147 1.73 147 1.73 28.5 3.00 32.6 2.62 102 2.50 132 1.93 28.5 3.00 32.6 2.62 147 1.73 147 1.73 102 2.50 132 1.93 147 1.73 147 1.73 147 1.73 147 1.73 102 2.50 132 1.93 28.5 3.00 32.6 2.62 147 1.73 147 1.73 102 2.50 132 1.93 147 1.73 147 1.73 102 2.50 132 1.93 102 2.50 132 1.93 147 1.73 147 1.73 102 2.50 132 1.93 147 1.73 147 1.73 45.5 4.89 60.4 3.69 102 2.50 132 1.93 147 1.73 147 1.73 45.5 4.89 60.4 3.69 147 1.73 147 1.73 45.5 4.89 60.4 3.69 147 1.73 147 1.73 45.5 4.89 60.4 3.69 147 147 1.73 147 1.73 147 1.73 45.5 4.89 60.4 3.69 45.5 4.89 60.4 3.69 147 1.73 1.73 147 1.73 45.5 4.89 60.4 3.69 45.5 4.89 60.4 3.69 45.5 4.89 60.4 3.69 45.5 4.89 60.4 3.69 45.5 4.89 60.4 3.69 45.5 4.89 60.4 3.69 45.5 4.89 60.4 3.69 5.73 3.89 5.46 4.08 45.5 4.89 60.4 3.69 45.5 4.89 60.4 3.69 5.73 3.89 5.46 4.08 5.73 3.89 5.46 4.08 5.73 3.89 5.46 4.08 5.73 3.89 5.46 4.08 5.73 3.89 5.46 4.08 5.73 3.89 5.46 4.08 5.73 3.89 5.46 4.08 5.73 3.89 5.46 4.08 5.73 3.89 5.46 4.08 5.73 3.89 5.46 4.08 5.73 3.89 5.46 4.08 5.73 3.89 3.89 5.46 5.46 4.08 4.08 5.73 5.73 3.89 5.46 4.08 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 7.26 3.84 8.59 3.24 14.8 5.58 7.12 11.6 7.12 11.6 14.8 5.58 7.12 11.6 14.8 5.58 7.12 11.6 14.8 5.58 7.12 11.6 14.8 5.58 7.12 11.6 14.8 5.58 7.12 11.6 14.8 5.58 7.12 11.6 14.8 5.58 7.12 11.6 14.8 5.58 7.12 11.6 14.8 5.58 7.12 11.6 14.8 7.12 11.6 14.8 5.58 145 3.00 >145 -5.58 7.12 11.6 14.8 5.58 7.12 11.6 14.8 5.58 7.12 11.6 14.8 5.58 145 3.00 >145 145 3.00 >145 145 3.00 >145 145 3.00 >145 145 3.00 >145 ---145 3.00 >145 145 3.00 >145 145 3.00 >145 145 3.00 >145 145 3.00 >145 --- 145 3.00 >145 2.67 5.40 2.84 5.08 145 3.00 >145 145 3.00 >145 --145 3.00 >145 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 2.67 5.40 2.84 5.08 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 1.61 5.74 1.85 4.97 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 11.9 4.62 12.3 4.46 >47.9 >47.9 >47.9 >47.9 >47.9 --->47.9 --->47.9 >47.9 >47.9 >47.9 >47.9 --- >47.9 --- >47.9 >47.9 >47.9 >47.9 >47.9 >47.9 >47.9 >47.9 >47.9 -->47.9 -->47.9 >47.9 43.6 3.83 45.7 3.65 >47.9 >47.9 >47.9 -->47.9 -->47.9 >47.9 43.6 3.83 45.7 3.65 43.6 3.83 45.7 3.65 43.6 3.83 45.7 3.65 43.6 3.83 45.7 3.65 43.6 3.83 45.7 3.65 43.6 3.83 45.7 3.65 43.6 3.83 45.7 3.65 43.6 3.83 45.7 3.65 43.6 3.83 45.7 3.65 43.6 3.83 45.7 3.65 43.6 3.83 45.7 3.65 >144 43.6 3.83 45.7 3.65 43.6 3.83 45.7 3.65 >144 43.6 3.83 45.7 3.65 >144 >144 >144 ---- >144 ---- >144 >144 >144 >144 >144 >144 >144 >144 >144 ->144 ->144 >144 >144 >144 >144 ->144 -->144 >144 >144 >144 -->144 --->144 >144 >144 109 3.05 >144 109 3.05 >144 109 3.05 >144 ---- 109 3.05 >144 109 3.05 >144 109 3.05 >144 109 3.05 >144 109 3.05 >144 -109 3.05 >144 109 3.05 >144 109 3.05 >144 -109 3.05 >144 109 3.05 >144 109 3.05 >144 ---109 3.05 >144 99.3 4.55 >143 99.3 4.55 >143 99.3 4.55 >143 99.3 4.55 >143 ---- 99.3 4.55 >143 99.3 4.55 >143 99.3 4.55 >143 99.3 4.55 >143 99.3 4.55 >143 99.3 4.55 >143 99.3 4.55 >143 -99.3 4.55 >143 99.3 4.55 >143 99.3 4.55 >143 ---99.3 4.55 >143 44.2 1.13 >47.8 44.2 1.13 >47.8 >47.8 44.2 1.13 >47.8 44.2 1.13 ---- 44.2 1.13 >47.8 44.2 1.13 >47.8 44.2 1.13 >47.8 --44.2 1.13 >47.8 44.2 1.13 >47.8 44.2 1.13 >47.8 44.2 1.13 >47.8 -44.2 1.13 >47.8 44.2 1.13 >47.8 44.2 1.13 >47.8 ---44.2 1.13 >47.8 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 19.4 7.35 27.6 5.18 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 5.65 82.2 3.73 54.2 129 2.72 >143 - 129 5.65 2.72 82.2 >143 3.73 54.2 5.65 82.2 3.73

BrCr Strain BrCr Strain Strain BrCr BrCr a Strain BrCr Strain 50 SI BrCr IC 50 aaa Strain BrCr Strain 50 a BrCr Strain IC 50 SI a IC 50 SI BrCr Strain IC 50 SI BrCr IC 50 aaa Strain SI 15.6 IC 50 SI BrCr Strain 9.45 a IC 50 SI BrCr Strain aStrain BrCr IC 50 SI Strain 9.45 15.6 BrCr IC 50BrCr SI 9.45 15.6 aa Strain 50 9.45 15.6 IC 50 SI 9.45 15.6 35.4 2.41 9.45 15.6 IC 50aaa SI 9.45 15.6 35.4 2.41 a IC SI IC 5050 SI 9.45 15.6 a IC 50 SI 35.4 2.41 9.45 15.6 IC50 15.6 35.4 2.41 35.4 2.41 9.45 135 1.87 SI 35.4 2.41 9.45 15.6 35.4 2.41 9.45 15.6 135 1.87 9.45 15.6 35.4 2.41 9.45 15.6 135 1.87 35.4 2.41 135 1.87 135 1.87 35.4 2.41 >147 - 15.6 9.45 135 1.87 35.4 2.41 135 1.87 35.4 2.41 >147 35.4 2.41 135 1.87 35.4 2.41 >147 ---- 2.41 135 1.87 >147 >147 135 35.4 1.87 >147 135 1.87 >147 135 1.87 135 >147 135135 1.87 1.87 >147 32.3 6.90 >147 --- 1.87 32.3 6.90 >147 32.3 6.90 >147 32.3 6.90 >147 >147 32.3 6.90 32.3 >147 6.90 32.3 6.90 32.3 6.90 32.3 6.90 32.3 6.90 32.3 6.90 32.3 6.90 32.3 2.0432.3 6.90 10.9 6.90 32.3 6.90 2.04 10.9 2.04 10.9 2.04 10.9 2.04 10.9 2.04 10.9 2.04 10.9 2.04 10.9 2.04 10.9 2.04 10.9 2.04 10.9 2.04 10.9 2.04 10.9 10.9 2.04 2.04 10.9 4.26 6.54 4.26 6.54 4.26 6.54 4.26 6.54 4.26 6.54 4.26 6.54 4.26 6.54 4.26 6.54 4.26 6.54 4.26 6.54 4.26 6.54 4.26 6.54 4.26 6.54 4.26 4.26 6.54 6.54 8.52 9.72 8.52 9.72 8.52 9.72 8.52 9.72 8.52 9.72 8.52 9.72 8.52 9.72 8.52 9.72 8.52 9.72 8.52 9.72 8.52 8.52 9.72 145 3.00 9.72 8.52 9.72 8.52 9.72 8.52 9.72 145 3.00 145 3.00 145 3.00 145 3.00 145 145 3.00 145145 3.00 3.00 3.00 145 3.00 145 3.00 145 3.00 2.23 6.47 145 3.00 145 3.00 145 3.00 2.23 6.47 2.23 6.47 2.23 6.47 2.23 6.47 2.23 6.47 2.23 6.47 2.232.23 6.47 6.47 6.47 2.23 2.23 6.47 2.23 6.47 2.23 6.47 2.23 2.23 6.47 6.47 2.21 4.17 2.21 4.17 2.21 4.17 2.21 4.17 2.21 4.17 2.21 4.17 2.21 4.17 2.212.21 4.17 4.17 4.17 2.21 2.21 4.17 2.21 4.17 2.21 4.17 2.21 2.21 4.17 4.17 10.6 5.18 10.6 5.18 10.6 5.18 10.6 5.18 10.6 5.18 10.6 5.18 10.6 5.18 10.6 5.18 10.610.6 5.18 5.18 5.18 10.6 10.6 5.18 10.6 5.18 10.6 10.6 5.18 47.9 5.18 2.59 47.9 2.59 47.9 2.59 47.9 47.9 2.59 47.9 2.59 47.9 2.59 47.9 2.59 47.9 2.59 2.59 47.9 2.59 47.9 2.59 47.9 2.59 33.6 2.59 4.97 47.9 2.59 47.9 47.9 2.59 33.6 4.97 33.6 33.6 4.97 33.6 4.97 33.6 4.97 4.97 33.6 4.97 33.6 4.97 33.6 4.97 33.6 4.97 33.6 4.97 33.6 4.97 >144 33.6 4.97 33.6 33.6 4.97 4.97 >144 >144 >144 >144 ---->144 >144 >144 >144 >144 ->144 >144 >144 >144 --->144 144 2.32 144144 2.32 2.32 2.32 144 144 2.32 144 2.32 144 2.32 144 2.32 144 2.32 144 2.32 144 2.32 144 2.32 144 2.32 144 2.32 144 2.32 81.1 5.57 81.181.1 5.57 5.57 5.57 81.1 81.1 5.57 81.1 5.57 81.1 5.57 81.1 5.57 81.1 5.57 81.1 5.57 81.1 5.57 81.1 5.57 81.1 5.57 81.1 81.1 5.57 44.244.2 5.57 1.13 1.13 44.2 1.13 44.2 1.13 44.2 1.13 44.2 1.13 44.2 1.13 44.2 1.13 44.2 1.13 44.2 1.13 44.2 1.13 44.2 1.13 44.2 1.13 44.2 44.2 1.13 1.13 17.117.1 8.36 8.36 8.36 17.1 17.1 8.36 17.1 8.36 17.1 8.36 17.1 8.36 17.1 8.36 17.1 8.36 17.1 8.36 17.1 8.36 17.1 8.36 17.1 8.36 17.1 17.1 8.36 8.36 of 16 66.066.0 54.64 4.64 66.0 4.64 66.0 4.64 66.0 4.64 66.0 4.64 66.0 4.64 66.0 4.64 66.0 4.64 66.0 4.64 66.0 4.64 66.0 4.64 66.0 4.64 66.0 >143 >1434.64 66.0 4.64

5a

250

130

1.93

109

2.29

144

1.74

125

2.01

5b

83.3

41.3

2.02

34.2

2.44

69.3

1.20

34.2

2.44

5c

248

93.7

2.65

101

2.46

123

2.01

44.1

5.62

5d

41.0

>15.9

-

9.01

4.55

12.0

3.41

>15.9

-

Molecules 2018, 23, x Molecules 2018, 23,23, Molecules 2018, x Molecules 2018, 23,2084 x

4g 4g 4g 4g

5 of 16

of 16 55 of 16 of 16 16 55 of of 16 16 55 of

Molecules 2018, 2018, 23, 23, x x Molecules Molecules 2018, 23, x

351 351 351 351

>143 >143 >143 >143

5a 5a 5a5a 5a

250 250 250 250 250

130 130 130 130 130

5b 5b 5b 5b 5b

83.3 83.3 83.3 83.3 83.3

5c 5c 5c 5c5c 5c

129 2.72 Table 1.129 Cont. 2.72 --129 2.72 129

2.72

>143 >143 >143 >143

1.93 1.93 1.93 1.93

109 109 109 109 109

2.29 2.29 2.29 2.29 2.29

144 144 144144 144

1.74 1.74 1.74 1.74 1.74

125 2.01 125 2.01 125125 2.01 2.01 2.01 125

41.3 41.3 41.3 41.3 41.3

2.02 2.02 2.02

34.2 34.2 34.2 34.2 34.2

2.44 2.44 2.44 2.44 2.44

69.3 69.3 69.3 69.3 69.3

1.20 1.20 1.20 1.20 1.20

34.2 2.44 34.2 2.44 34.234.2 2.44 2.44 2.44 34.2

248 248 248 248 248 248

93.7 93.7 93.7 93.7 93.7 93.7

2.65 2.65 2.65 2.65

101 101 101 101 101 101

2.46 2.46 2.46 2.46 2.46 2.46

123 123 123 123123 123

2.01 2.01 2.01 2.01 2.01 2.01

44.1 44.1 44.1 44.144.1 44.1

5d 5d 5d 5d 5d

41.0 41.0 41.0 41.0 41.0

>15.9 >15.9 >15.9 >15.9 >15.9

---

9.01 9.01 9.01 9.01 9.01

4.55 4.55 4.55 4.55 4.55

12.0 12.0 12.0 12.0 12.0

3.41 3.41 3.41 3.41 3.41

>15.9 >15.9 -->15.9 >15.9 >15.9 -

5e5e 5e 5e 5e

27.6 27.6 27.6 27.6 27.6 27.6

10.4 10.4 10.4 10.4 10.4 10.4

2.65 2.65 2.65 2.65 2.65

8.69 8.69 8.69 8.69 8.69 8.69

3.18 3.18 3.18 3.18 3.18 3.18

9.13 9.13 9.13 9.13 9.13 9.13

3.03 3.03 3.03 3.03 3.03 3.03

9.20 3.01 9.20 3.01 9.20 3.01 9.209.20 3.01 3.01 3.01 9.20

5f5f 5f 5f 5f

143 143 143 143 143

40.4 40.4 40.4 40.4 40.4

3.54 3.54 3.54 3.54 3.54

27.0 27.0 27.0 27.0 27.0

5.36 5.36 5.36 5.36 5.36

68.4 68.4 68.4 68.4 68.4

2.09 2.09 2.09 2.09 2.09

25.425.4 5.63 5.63 5.63 25.4 25.4 5.63 25.4 5.63

-

---

>143 >143 >143 >143

---

5.62 5.62 5.62 5.62 5.62 5.62 -

265 143 1.85 51.3 5.16 86.7 3.05 82.882.8 3.19 3.19 5g5g 265 143 51.3 5.16 86.7 3.05 1.85 5g 265 143 1.85 51.3 5.16 86.7 3.05 82.8 3.19 5g 265 143 1.85 51.3 5.16 86.7 3.05 82.8 3.19 5g 265 143 1.85 51.3 5.16 86.7 3.05 82.8 3.19 a a The aa The 50 (median 50 values unit of µM was used to measure TC (median toxic concentration) and IC values which are calculated unit of μM was used to measure TC 50 toxic concentration) and IC 50 which are a 50 50 50 (median toxic concentration) and 50 50 values which are a The unit of μM was used to measure TC 50 50 IC 50 aa The Theonunit unit of μM was was used totarget measure TC50 50 (median (median toxic concentration) concentration) and IC50 50 values values which are are μM used to measure TC toxic and IC which based the of organic part the compounds. The average values three batches were shown in this table calculated based on on theoforganic organic part of the the target target compounds. The of average values of of three batches calculated based the part of compounds. The average values three batches b calculated based on the the as organic part of the target compounds. The average average values values of of three three batches batches calculated based on organic of the target compounds. The bpart (n = 3). SI was calculated the ratio of TC and IC . b 50 50 50 and IC50 50. b were shown in this table (n = 3). b SI was calculated as the ratio of TC50 50 50 b 50 and IC50 50 SI was calculated as the ratio of TC 50 were shown in this table (n 3). SI was was calculated calculated as as the the ratio ratio of of TC TC50 50 and and IC IC50 50... were shown shown in in this this table table (n (n === 3). 3). bb SI were

In the second round of SAR study, the methylenedioxy ring was opened and substituent on In the the second round of of SAR SAR 1, study, the JTH methylenedioxy ring against was opened opened and substituent substituent on all of First, thesecond key intermediates 3, and were screened H strain of EV71, and In round study, the methylenedioxy ring was and on In the second round of SAR study, the methylenedioxy ring was opened Only and substituent on position 9 of ring D was attached, by which seven analogues 4a–g were created. compound 4e position 9 of ring D was attached, by which seven analogues 4a–g were created. Only compound 4e on the position of decreased ring D D was was attached, attached, by which which seven seven analogues 4a–gSAR wereanalysis created. Only Only compound 4e them showed activity compared withanalogues that of BBR. was then focused position 99 of ring by 4a–g were created. compound 4e with a 9-m-methoxybenzyloxycarbonylmethylenoxy moiety displayed a comparable activity to BBR. with a 9-m-methoxybenzyloxycarbonylmethylenoxy moiety displayed a comparable activity to BBR. with aa 9-m-methoxybenzyloxycarbonylmethylenoxy 9-m-methoxybenzyloxycarbonylmethylenoxy moiety displayed comparable activity activity to BBR. BBR. influence of substitutions on position 9 of ring D, moiety and seven etheraaderivatives (2a–g) and two ester with displayed comparable to Meanwhile, while methylenedioxy ring was opened and the substituent was switched to position Meanwhile, while while methylenedioxy methylenedioxy ring ring was was opened opened and and the the substituent substituent was was switched switched to to position position 33 3 Meanwhile, analogues (2h and 2i) were prepared and tested, respectively. As described in the table, among Meanwhile, while methylenedioxy ring was and opened and theAmong substituent was switched5e to gave position 3 them, of ring A, compounds 5a–g were prepared screened. them, compound ideal of ring A, compounds 5a–g were prepared and screened. Among them, compound 5e gave ideal of ring ring A, compounds compounds 5a–gdifferent were prepared prepared and screened. screened. Among them, them, compoundsatisfactory 5e gave gave ideal ideal of A, 5a–g were and Among compound 5e ether compounds 2d–gwith with substituents on the benzene ring displayed potencies activities compared BBR as well as high cytotoxicity. The inhibitory activity of compounds activities compared compared with with BBR BBR as as well well as as high high cytotoxicity. cytotoxicity. The The inhibitory inhibitory activity activity of of compounds compounds activities activities compared with BBR as well as high cytotoxicity. The inhibitory activity of compounds with IC values of 12.4 and 15.8 µM, especially compound 2d that exhibited lower cytotoxicity and 5a–d, 50 5f and and 5g 5g was was partially partially or or completely completely lost. lost. 5a–d, 5f 5a–d, 5f and 5g was partially or completely lost. a betterNext, SI value of 6.7, similar to that of BBR. Meanwhile, the against carboxylic acid form of compound 2d the anti-EV71 anti-EV71 activities of all all the target target compounds JS-52, SHZH98 and BrCr Next, the the activities of the compounds against JS-52, JS-52, SHZH98 and BrCr Next, anti-EV71 activities of allantivirus the targetpotency compounds against SHZH98 and BrCr (2d-a) exhibited a complete loss on the which indicated that compound 2d might strains were also tested respectively. As shown in Table 1, the screening results were almost strains were were also also tested tested respectively. respectively. As As shown shown in in Table Table 1, 1, the the screening screening results results were were almost almost strains consistent with that thatits oforiginal H strain. strain.form. TheseTwo results indicated that appropriate appropriate substituents ondecreased position 99activity, exert the activity on ester derivatives 2h and 2i gave obviouson consistent with of H These results indicated that substituents position consistent with that of H strain. These results indicated that appropriate substituents on position 9 and methylenedioxy on ring ring A A might might be beneficial beneficial for the the on antivirus potency. Among them, for the Theand results hinted that introducing a suitable ether substituent position 9 mightAmong be beneficial and methylenedioxy on be for antivirus potency. Among them, methylenedioxy on ring satisfactory A might be beneficial for the antivirus potency. them, compound 2d exhibited most potency against all tested genotype EV71 strains as well compound 2dEV71. exhibited most most satisfactory satisfactory potency potency against against all all tested tested genotype genotype EV71 EV71 strains strains as as well well ability against2d compound exhibited as high SI values, which indicated that compound 2d exhibited a broad-spectrum anti-EV71 activity, as In high SI second values, which which indicated that compound 2d exhibited exhibited aa broad-spectrum broad-spectrum anti-EV71 activity, the roundindicated of SARthat study, the methylenedioxy ring was opened and substituent on as high SI values, compound 2d anti-EV71 activity, and was was selected selected as as the the representative representative compound compound for for further further investigation. investigation. and position 9 of ring D attached, by compound which seven 4a–g were created. Only compound 4e and was selected aswas the representative for analogues further investigation. Furthermore, microscopy microscopy and and crystal crystal violet violet staining staining was was used used to to further further investigation investigation the the Furthermore, Furthermore, microscopy and crystal violet staining was used to further investigation the with a 9-m-methoxybenzyloxycarbonylmethylenoxy moiety displayed a comparable activity to BBR. anti-EV71 effect of 2d with CPE (cytopathic effect) assay [4]. As shown in Figure 2A,B, the anti-EV71 effect effect of of 2d 2d with with CPE CPE (cytopathic (cytopathic effect) effect) assay assay [4]. [4]. As As shown shown in in Figure Figure 2A,B, 2A,B, the the anti-EV71 Meanwhile, ring demonstrated was opened and was switched anti-EV71 while effect methylenedioxy of 2d 2d was was visually visually that the it substituent could significantly significantly reduce to theposition anti-EV71 effect of demonstrated that that it could reduce the anti-EV71 effect of 2d5a–g waswere visually demonstrated it could significantly reduce the 3 of ring A, compounds prepared and screened. Among them, compound 5e gave ideal EV71-induced CPE at the concentration of 20 μM, better than that of BBR. EV71-induced CPE CPE at at the the concentration concentration of of 20 20 μM, μM, better better than than that that of of BBR. BBR. EV71-induced

activities compared with BBR as well as high cytotoxicity. The inhibitory activity of compounds 5a–d, 2.2.2.5g Compound 2d Inhibits Inhibits EV71 Replication Replication in Both Both RNA RNA and and Protein Protein Level Level 5f 2.2.2. and was partially or completely lost. in 2.2.2. Compound 2d EV71 Compound 2d Inhibits EV71 Replication in Both RNA and Protein Level Next, the anti-EV71 of all the target the compounds against JS-52, activity SHZH98ofand To further further confirmactivities the anti-EV71 anti-EV71 activity, VP1 down-regulatory down-regulatory 2d BrCr was strains To confirm the activity, the VP1 VP1 activity of of 2d 2d was To further confirm the anti-EV71 activity, the down-regulatory activity was examined in Vero cells infected with H strain of EV71, in comparison with that of BBR. As shown in were also tested respectively. As shown the screening were almost examined in Vero Vero cells infected infected with with H strain straininof ofTable EV71, 1, in comparison comparison withresults that of of BBR. BBR. As As shownconsistent in examined in cells H EV71, in with that shown in Figure 2C, compound 2d significantly reduced VP1 RNA expression in a dose-dependently way, with that2C, of compound H strain. 2dThese resultsreduced indicated on position Figure significantly VP1that RNAappropriate expression in insubstituents dose-dependently way, 9 and Figure 2C,activity compound 2dwas significantly reduced VP1 RNA expression aatranscription-quantitative dose-dependently way, and the of 2d better than that of BBR in the reverse and the activity of 2d was better than that of BBR in the reverse transcription-quantitative methylenedioxy on ring A might be beneficial for the antivirus potency. Among them, compound 2d and the the activity activity of of 2d 2d was was better better than than that that of of BBR BBR in in the the reverse reverse transcription-quantitative transcription-quantitative and polymerase chain reaction (RT-qPCR) assay. Accordingly, VP1 capsid protein expression was also polymerase chain reaction (RT-qPCR) assay. Accordingly, VP1 capsid protein expression was also polymerase chain reaction potency (RT-qPCR) assay. all Accordingly, VP1 capsid capsid protein expression was also exhibited mostchain satisfactory against tested genotype EV71protein strainsexpression as well aswas high SI values, polymerase reaction (RT-qPCR) assay. Accordingly, VP1 also analyzed to examine its effect on EV71 biological synthesis. As depicted in Figure 2D, compound 2d analyzed to examine examine its effect effect on on2d EV71 biological synthesis. As As depicted depicted in Figure Figure 2D, compound compound 2d analyzed to its EV71 biological synthesis. in 2D, 2d which indicated that compound exhibited a broad-spectrum anti-EV71 activity, and was selected as analyzed to examine its effect on the EV71 biologicalof synthesis. As depicted in Figure 2D, compound 2d could obviously down-regulate expression VP1 protein in dose-dependent manner, and could obviously obviously down-regulate down-regulate the the expression expression of of VP1 VP1 protein protein in in aa a dose-dependent dose-dependent manner, manner, and and could thecould representative compound for further investigation. obviously down-regulate the expression of that VP1ofprotein in aconcentration dose-dependent manner, and compound 2d exhibited better antiviral effect than BBR at the of 40 μM. compound 2d 2d exhibited exhibited better better antiviral antiviral effect effect than than that that of of BBR BBR at at the the concentration concentration of of 40 40 μM. μM. compound compound 2d exhibited better antiviral effect than thatstaining of BBR atwas the concentration of 40 μM. Furthermore, microscopy and crystal violet used to further investigation the anti-EV71 effect of 2d with CPE (cytopathic effect) assay [4]. As shown in Figure 2A,B, the anti-EV71 effect of 2d was visually demonstrated that it could significantly reduce the EV71-induced CPE at the concentration of 20 µM, better than that of BBR.

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anti-EV71 effect effect of of compound compound 2d 2d in Vero cells with BBR as a positive Figure 2. The anti-EV71 positive control. (A) 2d reduced the ×40); (B) (B) 2d 2d reduced reduced the the reduced the EV71-induced EV71-induced CPE. CPE. Cells Cells were were examined examined using using aa microscopy microscopy ((×40); EV71-induced CPE. (C) 2d2d reduced thethe expression of EV71-induced CPE. Cells Cellswere wereexamined examinedusing usingcrystal crystalviolet violetstaining; staining; (C) reduced expression EV71 VP1 RNA by one-step qRT-PCR assay. ** p < 0.001 * p < 0.05; (D) 2d reduced the expression of of EV71 VP1 RNA by one-step qRT-PCR assay. ** p < 0.001 * p < 0.05; (D) 2d reduced the expression EV71 VP1 protein by by western blot assay. of EV71 VP1 protein western blot assay.

2.2.2. Compound 2d Inhibits EV71 Replication in Both RNA and Protein Level 2.3. Compound 2d Inhibits the Phosphorylation of MEK/ERK To further confirm theMEK/ERK anti-EV71 signaling activity, the VP1 down-regulatory activity of 2d examined The activation of the pathway has been certified to play anwas essential role infected H strain ofinEV71, in comparison with that BBR.first As shown in Figure 2C, in Vero EV71cells life cycle andwith pathogenesis various cell types [15–22]. Weofhave confirmed that BBR compound 2dthe significantly reducedofVP1 RNA expression dose-dependently and whether the activity could inhibit phosphorylation MEK/ERK induced in byaEV71 infection [4]. way, To learn or of 2d better than of BBR in the reverse transcription-quantitative chain reaction not 2dwas would still act that through MEK/ERK pathway as its parent BBR does,polymerase the inhibition experiment (RT-qPCR) VP1 capsid protein expression was alsoasanalyzed to examine its effect of EV71 viaassay. this Accordingly, signaling pathway was done with 2d using BBR the reference. As shown in on EV713,biological synthesis. As depicted in Figure 2D,the compound 2d could obviously down-regulate Figure compound 2d could significantly inhibit phosphorylation of both MEK and ERK the expression of VP1 protein so in aasdose-dependent manner, and compound 2d exhibited antiviral induced by EV71 infection, to attenuate the activation of MEK/ERK signalingbetter pathway. We effectconclude than that that of BBR at the concentration of 40the µM.replication of EV71 through down-regulating can compound 2d inhibited MEK/ERK signaling pathway. 2.3. Compound 2d Inhibits the Phosphorylation of MEK/ERK The activation of the MEK/ERK signaling pathway has been certified to play an essential role in EV71 life cycle and pathogenesis in various cell types [15–22]. We have first confirmed that BBR could inhibit the phosphorylation of MEK/ERK induced by EV71 infection [4]. To learn whether or not 2d would still act through MEK/ERK pathway as its parent BBR does, the inhibition experiment of EV71 via this signaling pathway was done with 2d using BBR as the reference. As shown in Figure 3, compound 2d could significantly inhibit the phosphorylation of both MEK and ERK induced by EV71 infection, so as to attenuate the activation of MEK/ERK signaling pathway. We can conclude that compound 2d inhibited the replication of EV71 through down-regulating MEK/ERK signaling pathway.

Figure 3. Compound 2d inhibits the phosphorylation of MEK/ERK with BBR as a control. Vero cells (9 × 105 cells/well) were plated into 6-well culture plates and were mock-infected or infected with EV71 (H, MOI = 0.1) for 1 h. The cells were then treated with compound 2d for 24 h and were harvested for western blot assay.

could inhibit the phosphorylation of MEK/ERK induced by EV71 infection [4]. To learn whether or not 2d would still act through MEK/ERK pathway as its parent BBR does, the inhibition experiment of EV71 via this signaling pathway was done with 2d using BBR as the reference. As shown in Figure 3, compound 2d could significantly inhibit the phosphorylation of both MEK and ERK induced by EV71 infection, so as to attenuate the activation of MEK/ERK signaling pathway. We can conclude that compound 2d inhibited the replication of EV71 through down-regulating Molecules 2018, 23, 2084 MEK/ERK signaling pathway.

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Figure 3. Compound 2d inhibits thethe phosphorylation ofMEK/ERK MEK/ERK as a control. Vero cells Figure 3. Compound 2d inhibits phosphorylation of withwith BBR BBR as a control. Vero cells were platedinto into6-well 6-well culture culture plates and were mock-infected or infected with with × 105 cells/well) (9 × 105(9cells/well) were plated plates and were mock-infected or infected EV71 MOIfor = 0.1) 1 h. Thewere cells then weretreated then treated compound 2d 24 for h24and h and were EV71 (H, MOI(H, = 0.1) 1 h.for The cells with with compound 2d for were harvested harvested for western blot assay. for western blot assay. Molecules 2018, 23, x 2.4. Compound 2d Inhibits EV71-Induced Autophagy

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2.4. Compound Inhibits EV71-Induced EV71 infection2dtriggers autophagy,Autophagy which provides a support for EV71 replication [23]. Both JNK and PI3KIII proteins as upstream activators, play important rolesreplication in regulating autophagy, EV71 infection triggers autophagy, which provides a support for EV71 [23]. Both JNK andof PI3KIII proteins as upstream important roles in regulating autophagy, and and inhibition corresponding signalingactivators, pathwayplay could down-regulate autophagy [24]. Otherwise, of corresponding signaling pathway could autophagy down-regulate autophagy [24]. Otherwise, AKT, asinhibition an inhibitor of autophagy factor, could inhibit with its activation [25]. To further AKT, as an inhibitor of autophagy factor, could inhibit autophagy with its activation [25]. To understand whether 2d still affected autophagy after structure modification, the inhibition of activation further understand whether 2d still affected autophagy after structure modification, the inhibition AKT andofphosphorylation of JNK and PI3KIII proteins were also carried out using BBR as a reference [4]. activation AKT and phosphorylation of JNK and PI3KIII proteins were also carried out using As shown in Figure 4, compound 2d could activate AKT and the phosphorylation JNK and BBR as a reference [4]. As shown in Figure 4, compound 2dinhibit could activate AKT and inhibitofthe PI3KIII proteins similarofto BBR reported before. phosphorylation JNK andasPI3KIII proteins similar to BBR as reported before.

4. Compound 2d inhibits EV71-induced autophagy. Vero were mock-infectedororinfected Figure 4.Figure Compound 2d inhibits EV71-induced autophagy. Vero cellscells were mock-infected infected with EV71 (H, MOI = 0.1) for 1 h. The cells were then treated with compound 2d and BBR with EV71 (H, MOI = 0.1) for 1 h. The cells were then treated with compound 2d and BBR for 24 h. for 24 h. The cells were harvested and proteins were examined by western blot. The cells were harvested and proteins were examined by western blot.

Accordingly, we found that the lapidated LC3II, a marker for autophagy, decreased in the presence of compound 2d, while compound 2d could not affect the expression of Beclin-1, similar to that of BBR. Therefore, compound 2d exerted a potent anti-EV71 effect through the same mechanism with parent BBR after the structure modification. Collectively, as depicted in the cartoon chart (Figure 5), compound 2d might exert a potent anti-EV71 activity through inhibiting the activation of MEK/ERK pathway and suppressing EV71-induced autophagy by activating AKT and inhibiting the phosphorylation of JNK and PI3KIII. As a result, compound 2d owned a potent

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Accordingly, we found that the lapidated LC3II, a marker for autophagy, decreased in the presence of compound 2d, while compound 2d could not affect the expression of Beclin-1, similar to that of BBR. Therefore, compound 2d exerted a potent anti-EV71 effect through the same mechanism with parent BBR after the structure modification. Collectively, as depicted in the cartoon chart (Figure 5), compound 2d might exert a potent anti-EV71 activity through inhibiting the activation of MEK/ERK pathway and suppressing EV71-induced autophagy by activating AKT and inhibiting the phosphorylation of JNK and PI3KIII. As a result, compound 2d owned a potent anti-EV71 effect through the same action mode with Molecules 2018,parent 23, x BBR after the structure modification. 8 of 16

Figure 5. The cartoon chart of mechanism of compound 2d against EV71. Figure 5. The cartoon chart of mechanism of compound 2d against EV71.

3. Experimental Section 3. Experimental Section 3.1. Apparatus, Materials, and Analysis Reagents 3.1. Apparatus, Materials, and Analysis Reagents Melting points (mps) were obtained with a CXM-300 melting point apparatus (Shanghai Melting points (mps) were obtained with a CXM-300 melting point apparatus (Shanghai Changfang Optical Instrument Co., LTD., Shanghai, China) and are uncorrected. The 11H-NMR Changfang Optical Instrument Co., LTD., Shanghai, China) and are uncorrected. The H-NMR spectra was recorded on an Inova 500 or 600 MHz spectrometer (Varian, San Francisco, CA, USA) spectra was recorded on an Inova 500 or 600 MHz spectrometer (Varian, San Francisco, CA, USA) and 13C-NMR on an Avance III 400, 500 or 600 spectrometer (Bruker, Zürich, Switzerland) with and 13 C-NMR on an Avance III 400, 500 or 600 spectrometer (Bruker, Zürich, Switzerland) with Me4 Si as Me4Si as the internal standard, all the samples were dissolved in DMSO-d6 before testing. High the internal standard, all the samples were dissolved in DMSO-d6 before testing. High resolution mass resolution mass spectra (HRMS-ESI) data was recorded on an Autospec UItima-TOF mass spectra (HRMS-ESI) data was recorded on an Autospec UItima-TOF mass spectrometer (Micromass spectrometer (Micromass UK Ltd., Manchester, UK). Flash chromatography was performed on UK Ltd., Manchester, UK). Flash chromatography was performed on CombiflashRf 200 (Teledyne, CombiflashRf 200 (Teledyne, Lincoln, NE, USA), particle size 0.038 mm. Lincoln, NE, USA), particle size 0.038 mm. Vero cells were purchased from the American Type Culture Collection (ATCC, Rockefeller, Vero cells were purchased from the American Type Culture Collection (ATCC, Rockefeller, MD, MD, USA), and were cultured in Minimum Essential Medium (MEM) supplemented with 10% fetal USA), and were cultured in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine bovine serum (FBS) (GIBCO, Grand Island, NY, USA) and antibiotics (100 U/mL penicillin and 100 serum (FBS) (GIBCO, Grand Island, NY, USA) and antibiotics (100 U/mL penicillin and 100 mg/mL mg/mL streptomycin) at 37 °C in a 5% CO2 incubator. EV71 strain SHZH98 isolated from the throat streptomycin) at 37 ◦ C in a 5% CO2 incubator. EV71 strain SHZH98 isolated from the throat swab swab sample of an HFMD case occurring in 1998 in China was kindly provided by Dr. Qi Jin sample of an HFMD case occurring in 1998 in China was kindly provided by Dr. Qi Jin (Institute of (Institute of Pathogen Biology, Chinese Academy of Medical Science and Peking Union Medical Pathogen Biology, Chinese Academy of Medical Science and Peking Union Medical School, Beijing, School, Beijing, China), and JS-52 strain was a kind gift from Dr. Xiangzhong Ye (Beijing Wantai China), and JS-52 strain was a kind gift from Dr. Xiangzhong Ye (Beijing Wantai Biological Pharmacy Biological Pharmacy Enterprise Co., Ltd., Beijing, China). The EV71 BrCr and H strains were Enterprise Co., Ltd., Beijing, China). The EV71 BrCr and H strains were purchased from the ATCC. purchased from the ATCC. All of EV71 strains were passaged in Vero cells. All of EV71 strains were passaged in Vero cells. 3.2. Chemistry 3.2.1. General Procedure for the Synthesis of Compounds 2a–i BBR (1.86 g, 5 mmol) was heated at 195–210 °C for 10–15 min under vacuum (30–40 mmHg) to afford a black oil, which was acidified with ethanol/concentrated HCl (95:5). The solvent was removed by evaporation, the residue was collected and then purified by flash chromatography over silica gel using CH2Cl2/CH3OH as the gradient eluent, affording the title compound 1 (1.43 g, 80%) as a yellow solid. To a stirred solution of 1 (100 mg, 0.31 mmol) in anhydrous CH3CN or DMF,

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3.2. Chemistry 3.2.1. General Procedure for the Synthesis of Compounds 2a–i BBR (1.86 g, 5 mmol) was heated at 195–210 ◦ C for 10–15 min under vacuum (30–40 mmHg) to afford a black oil, which was acidified with ethanol/concentrated HCl (95:5). The solvent was removed by evaporation, the residue was collected and then purified by flash chromatography over silica gel using CH2 Cl2 /CH3 OH as the gradient eluent, affording the title compound 1 (1.43 g, 80%) as a yellow solid. To a stirred solution of 1 (100 mg, 0.31 mmol) in anhydrous CH3 CN or DMF, K2 CO3 (122 mg, 0.88 mmol) was added and heated to 70 ◦ C. Then the R1 Br or R2 Cl (2–4.8 eq) was added and stirred for 5–6 h. The mixture was cooled to precipitate completely, filtrated and then purified by flash chromatography over silica gel using CH2 Cl2 /CH3 OH as the gradient eluent to afford compounds 2a–i. 2,3-Methylenedioxy-9-ethyloxyformylmethylenoxy-10-methoxy protoberberine chloride (2a). Compound 1 (1.5 g, 4.2 mmol) was treated with ethyl bromoacetate (2.22 mL, 20 mmol) according to the general procedure to give the desired product 2a. Yield: 58%; yellow solid; m.p. 220–222 ◦ C; 1 H-NMR (400 MHz) δ 9.95 (s, 1H), 8.95 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.81 (s, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 5.08 (d, J = 4.0 Hz, 2H), 4.94 (t, J = 8.0 Hz, 2H), 4.18 (q, J = 16.0, 2H), 4.04 (s, 3H), 3.22 (t, J = 8.0 Hz, 2H), 1.21 (t, J = 8.0 Hz, 3H). 13 C-NMR (101 MHz) δ 169.2, 149.8, 149.2, 147.6, 145.6, 137.5, 132.8, 130.6, 126.6, 123.5, 120.3, 120.0, 108.3, 105.4, 102.0, 69.2, 69.1, 60.7, 57.1, 55.3, 51.8, 26.3, 14.0. HRMS: calcd. for C23 H22 ClNO6 [M]+ : 408.1442, found: 408.1444. 2,3-Methylenedioxy-9-isobutyloxyformylmethylenoxy-10-methoxy protoberberine chloride (2b). Compound 1 (1.5 g, 4.2 mmol) was treated with isobutyl bromoacetate (2.93 mL, 20 mmol) according to the general procedure to give the desired product 2b. Yield: 55%; yellow solid; m.p. 232–234 ◦ C; 1 H-NMR (400 MHz) δ 9.93 (s, 1H), 8.95 (s, 1H), 8.20 (d, J = 4.0 Hz, 1H), 7.97 (d, J = 4.0 Hz, 1H), 7.80 (s, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 4.97 (s, 2H), 4.94 (t, J = 4.0 Hz, 2H), 4.08 (d, J = 12.0 Hz, 1H), 4.04 (s, 2H), 3.31 (s, 3H), 3.21 (t, J = 4.0 Hz, 2H), 1.40 (t, J = 7.2 Hz, 6H). 13 C-NMR (101 MHz) δ 167.7, 149.8, 148.7, 147.6, 145.5, 141.5, 137.5, 132.9, 130.6, 126.8, 123.0, 120.9, 120.3, 120.0, 108.3, 105.4, 102.0, 81.6, 69.5, 61.8, 57.1, 55.3, 27.6 (2), 26.3. HRMS: calcd. for C25 H26 ClNO6 [M]+ : 436.1755, found: 436.1760. 2,3-Methylenedioxy-9-pivalylmethylenoxy-10-methoxy protoberberine chloride (2c). Compound 1 (357 mg, 1 mmol) was treated with 1-bromo-3,3-dimethyl-2-butanone (358.1 mg, 2 mmol) according to the general procedure to give the desired product 2c. Yield: 41%; yellow solid; m.p. 242–244 ◦ C; 1 H-NMR (500 MHz) δ 9.94 (s, 1H), 8.94 (s, 1H), 8.16 (d, J = 10.0 Hz, 1H), 7.95 (d, J = 10.0 Hz, 1H), 7.81 (s, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 5.51 (s, 2H), 4.95 (t, J = 5.0 Hz, 2H), 4.01 (s, 3H), 3.22 (t, J = 5.0 Hz, 2H), 1.15 (s, 9H). 13 C-NMR (126 MHz) δ 209.9, 149.7, 148.7, 147.6, 145.7, 141.9, 137.3, 132.8, 130.6, 126.5, 122.6, 121.0, 120.3, 119.9, 108.3, 105.3, 102.0, 73.1, 56.9, 55.3, 42.1, 26.3, 25.7 (3). HRMS: calcd. for C25 H26 ClNO5 [M]+ : 420.1806, found: 420.1807. 2,3-Methylenedioxy-9-benzyloxyformylmethylenoxy-10-methoxyprotoberberine chloride (2d). Compound 1 (1.5 g, 4.2 mmol) was treated with benzyl bromoacetate (3.17 mL, 20 mmol) according to the general procedure to give the desired product 2d. Yield: 58%; yellow solid; m.p. 206–208 ◦ C; 1 H-NMR (400 MHz) δ 9.90 (s, 1H), 8.92 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.80 (s, 1H), 7.34–7.29 (m, 5H), 7.09 (s, 1H), 6.18 (s, 2H), 5.19 (s, 2H), 5.16 (s, 2H), 4.84 (t, J = 4.0 Hz, 2H), 3.99 (s, 3H), 3.17 (t, J = 4.0 Hz, 2H). 13 C-NMR (101 MHz) δ 168.7, 149.8, 149.0, 147.6, 145.6, 141.3, 137.4, 135.3, 132.8, 130.5, 128.3 (2),128.1 (3), 126.6, 123.4, 121.1, 120.3, 120.0, 108.3, 105.3, 102.0, 69.2, 66.1, 57.0, 55.2, 26.2. HRMS: calcd. for C28 H24 ClNO6 [M]+ : 470.1598, found: 470.1598. 2,3-Methylenedioxy-9-carboxylmethylenoxy-10-methoxy protoberberine chloride (2d-a). Compound 2d (0.5 g, 1.1 mmol) was treated with concentrated hydrochloric acid (5.0 mL) at 50 ◦ C to give the desired product 2d-a. Yield: 78%; yellow solid; m.p. 232–234 ◦ C; 1 H-NMR (600 MHz) δ 13.12 (br, 1H), 9.95 (s, 1H), 8.94 (s, 1H), 8.20 (d, J = 6.0 Hz, 1H), 7.99 (d, J = 6.0 Hz, 1H), 7.80 (s, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 4.98 (s, 2H),

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4.93 (t, J = 6.0 Hz, 2H), 4.04 (s, 3H), 3.21 (t, J = 6.0 Hz, 2H). 13 C-NMR (151 MHz) δ 170.1, 149.8, 149.2, 147.6, 145.8, 141.7, 137.4, 132.9, 130.6, 126.7, 123.2, 121.2, 120.3, 120.0, 108.3, 105.4, 102.0, 69.1, 57.1, 55.3, 26.3. HRMS: calcd. for C21 H18 ClNO6 [M]+ : 380.1129, found: 380.1132. 2,3-Methylenedioxy-9-p-tolylformylmethylenoxy-10-methoxy protoberberine chloride (2e). Compound 1 (357 mg, 1 mmol) was treated with 2-bromo-40 -methyl acetophenone (426.1 mg, 2 mmol) according to the general procedure to give the desired product 2e. Yield: 42%; yellow solid; m.p. 219–221 ◦ C; 1 H-NMR (500 MHz) δ 10.01 (s, 1H), 8.96 (s, 1H), 8.18 (d, J = 5.0 Hz, 1H), 7.98 (d, J = 10.0 Hz, 1H), 7.82 (s, 1H), 7.60–7.58 (m, 1H), 7.52–7.47 (m, 2H), 7.29–7.27 (m, 1H), 7.11 (s, 1H), 6.19 (s, 2H), 5.89 (s, 2H), 4.96 (t, J = 5.0 Hz, 2H), 3.96 (s, 3H), 3.83 (s, 3H), 3.23 (t, J = 5.0 Hz, 2H). 13 C-NMR (126 MHz) δ 194.2, 159.4, 149.7, 148.9, 147.6, 145.7, 141.9, 137.4, 135.2, 132.9, 130.6, 130.1, 126.6, 122.9, 121.0, 120.3, 120.1, 120.0, 119.8, 112.2, 108.3, 105.4, 102.0, 74.8, 57.0, 55.3, 55.3, 26.3. HRMS: calcd. for C28 H24 ClNO5 [M]+ : 454.1649, found: 454.1653. 2,3-Methylenedioxy-9-m-methoxyphenylformylmethylenoxy-10-methoxy protoberberine chloride (2f). Compound 1 (357 mg, 1 mmol) was treated with 2-bromo-30 -methoxyacetophenone (458.1 mg, 2 mmol) according to the general procedure to give the desired product 2f. Yield: 41%; yellow solid; m.p. 214–216 ◦ C; 1 H-NMR (500 MHz) δ 10.02 (s, 1H), 8.95 (s, 1H), 8.17 (d, J = 10.0 Hz, 1H), 7.97 (d, J = 10.0 Hz, 1H), 7.89 (d, J = 10.0 Hz, 2H), 7.82 (s, 1H), 7.38 (d, J = 10.0 Hz, 2H), 7.11 (s, 1H), 6.18 (s, 2H), 5.87 (s, 2H), 4.95 (t, J = 5.0 Hz, 2H), 3.94 (s, 3H), 3.22 (t, J = 5.0 Hz, 2H), 2.40 (s, 3H). 13 C-NMR (126 MHz) δ 193.8, 149.7, 148.9, 147.6, 145.8, 144.4, 142.0, 137.4, 132.9, 131.4, 130.6, 129.4 (2), 127.7 (2), 126.6, 122.8, 121.0, 120.3, 120.0, 108.3, 105.3, 102.0, 74.6, 57.0, 55.3, 26.3, 21.2. HRMS: calcd. for C28 H24 ClNO6 [M]+ : 470.1598, found: 470.1598. 2,3-Methylenedioxy-9-p-trifloromethylphenylformylmethylenoxy-10-methoxy protoberberine chloride (2g). Compound 1 (357 mg, 1 mmol) was treated with 4-(trifluoromethyl)phenacyl bromide (534.1 mg, 2 mmol) according to the general procedure to give the desired product 2g. Yield: 40%; yellow solid; m.p. 238–240 ◦ C; 1 H-NMR (500 MHz) δ 10.02 (s, 1H), 8.97 (s, 1H), 8.20 (t, J = 10.0 Hz, 3H), 7.98 (t, J = 10.0 Hz, 3H), 7.82 (s, 1H), 7.11 (s, 1H), 6.19 (s, 2H), 5.93 (s, 2H), 4.96 (t, J = 5.0 Hz, 2H), 3.94 (s, 3H), 3.23 (t, J = 5.0 Hz, 2H). 13 C-NMR (126 MHz) δ 194.0, 149.8, 148.9, 147.6, 145.7, 141.7, 137.5, 137.2, 133.1, 132.9, 132.9, 130.6, 128.6 (3), 126.6, 125.8, 123.1, 120.9, 120.3, 120.0, 108.4, 105.4, 102.0, 75.0, 57.0, 55.3, 26.3. HRMS: calcd. for C28 H21 ClF3 NO5 [M]+ : 508.1366, found: 508.1366. 2,3-Methylenedioxy-9-benzyloxyformyloxy-10-methoxy protoberberine chloride (2h). Compound 1 (357 mg, 1 mmol) was treated with benzyl chloroformate (682.4 mg, 4 mmol) according to the general procedure to give the desired product 2h. Yield: 38%; yellow solid; m.p. 195–197 ◦ C; 1 H-NMR (400 MHz) δ 10.03 (s, 1H), 9.06 (s, 1H), 8.31 (d, J = 10.0 Hz, 1H), 8.24 (d, J = 15.0 Hz, 1H), 7.82 (s, 1H), 7.52–7.44 (m, 5H), 7.11 (s, 1H), 6.19 (s, 2H), 5.41 (s, 2H), 4.94 (t, J = 10.0 Hz, 2H), 4.02 (s, 3H), 3.22 (t, J = 10.0 Hz, 2H). 13 C-NMR (101 MHz) δ 151.5, 150.2, 150.0, 147.6, 144.2, 138.2, 134.6, 133.4, 132.7, 130.8, 128.6, 128.5 (2), 128.1 (2), 127.0, 125.9, 120.5(2), 120.2, 108.3, 105.4, 102.0, 70.7, 57.2, 55.3, 26.1. HRMS: calcd. for C27 H22 ClNO6 [M]+ : 456.1442, found: 454.1441. 2,3-Methylenedioxy-9-ethyloxyformyloxy-10-methoxy protoberberine chloride (2i). Compound 1 (1.5 g, 4.2 mmol) was treated with ethyl chloroformate (1.91 mL, 20 mmol) according to the general procedure to give the desired product 2i. Yield: 37%; yellow solid; m.p. 218–220 ◦ C; 1 H-NMR (400 MHz) δ 9.99 (s, 1H), 9.06 (s, 1H), 8.32 (d, J = 4.0 Hz, 1H), 8.24 (d, J = 4.0 Hz, 1H), 7.82 (s, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 4.95 (t, J = 4.0 Hz, 2H), 4.38 (q, J = 8.0 Hz, 2H), 4.08 (s, 3H), 3.22 (t, J = 4.0 Hz, 2H), 1.38 (t, J = 8.0 Hz, 3H). 13 C-NMR (101 MHz) δ 152.0, 150.9, 150.5, 148.2, 144.8, 138.7, 133.9, 133.3, 131.4, 127.5, 126.5, 121.1(2), 120.8, 108.9, 106.0, 102.6, 66.3, 57.8, 55.8, 26.6, 14.4. HRMS: calcd. for C22 H20 ClNO6 [M]+ : 394.1285, found: 394.1285.

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3.2.2. General Procedure for the Synthesis of Compounds 4a–g PMT (1.94 g, 5 mmol) was heated at 195–210 ◦ C for 10–15 min under vacuum (30–40 mmHg) to afford the black oil, which was acidified with ethanol/concentrated HCl (95:5). The solvent was removed by evaporation, the residue was collected and then purified by flash chromatography over silica gel using CH2 Cl2 /CH3 OH as the gradient eluent, affording the title compound 3 (1.6 g, 86%) as a yellow solid. To a stirred solution of compound 3 (100 mg, 0.30 mmol) in anhydrous CH3 CN or DMF, K2 CO3 (122 mg, 0.88 mmol) was added at room temperature. Then R1 Br or benzyl chloroformate (2–4 eq) was added and stirred for 0.5–1 h. The mixture was cooled to precipitate completely, filtrated and washed by CH2 Cl2 to afford compounds 4a–g. The final products were purified by flash chromatography over silica gel using CH2 Cl2 /CH3 OH as the gradient eluent. 2,3,10-Trimethoxy-9-ethyloxyformylmethylenoxy protoberberine chloride (4a). Compound 3 (373 mg, 1 mmol) was treated with ethyl bromoacetate (668 mg, 4 mmol) according to the general procedure to give the desired product 4a. Yield: 24%; yellow solid; m.p. 204–206 ◦ C; 1 H-NMR (500 MHz) δ 9.95 (s, 1H), 9.05 (s, 1H), 8.21 (d, J = 10.0 Hz, 1H), 8.03 (d, J = 10.0 Hz, 1H), 7.73 (s, 1H), 7.11 (s, 1H), 5.07 (s, 2H), 4.96 (t, J = 5.0 Hz, 2H), 4.18 (dd, J = 5.0 Hz, J = 10.0 Hz, 2H), 4.04 (s, 3H), 3.94 (s, 3H), 3.88 (s, 3H), 3.24 (t, J = 10.0 Hz, 2H), 1.21 (t, J = 10.0 Hz, 3H). 13 C-NMR (126 MHz) δ 168.8, 151.4, 148.9, 148.6, 145.7, 141.4, 137.7, 132.9, 128.5, 126.7, 123.2, 121.0, 119.7, 118.8, 111.2, 108.6, 69.2, 60.7, 57.0, 56.0, 55.8, 55.5, 25.9, 14.0. HRMS: calcd. for C24 H26 ClNO6 [M]+ : 424.1755, found: 424.1757. 2,3,10-Trimethoxy-9-pivalylmethylenoxy protoberberine chloride (4b). Compound 3 (500 mg, 1.34 mmol) was treated with 1-bromo-3,3-dimethyl-2-butanone (479.9 mg, 2.68 mmol) according to the general procedure to give the desired product 4b. Yield: 25%; yellow solid; m.p. 227–229 ◦ C; 1 H-NMR (500 MHz) δ 9.94 (s, 1H), 9.03 (s, 1H), 8.17 (d, J = 10.0 Hz, 1H), 7.98 (d, J = 10.0 Hz, 1H), 7.73 (s, 1H), 7.11 (s, 1H), 5.51 (s, 2H), 4.97 (t, J = 5.0 Hz, 2H), 4.02 (s, 3H), 3.94 (s, 3H), 3.88 (s, 3H), 3.24 (t, J = 5.0 Hz, 2H), 1.15 (s, 9H). 13 C-NMR (126 MHz) δ 209.9, 151.3, 148.6 (2), 145.7, 141.9, 137.5, 132.9, 128.5, 126.5, 122.5, 120.9, 119.6, 118.8, 111.1, 108.6, 73.1, 56.9, 56.0, 55.7, 55.4, 42.1, 25.9, 25.7 (3). HRMS: calcd. for C26 H30 ClNO5 [M]+ : 436.2119, found: 436.2119. 2,3,10-Trimethoxy-9-benzyloxyformylmethylenoxy protoberberine chloride (4c). Compound 3 (373 mg, 1 mmol) was treated with benzyl bromoacetate (916.2 mg, 4 mmol) according to the general procedure to give the desired product 4c. Yield: 29%; yellow solid; m.p. 196–198 ◦ C; 1 H-NMR (500 MHz) δ 9.90 (s, 1H), 9.01 (s, 1H), 8.20 (d, J = 9.2 Hz, 1H), 8.02 (d, J = 9.1 Hz, 1H), 7.71 (s, 1H), 7.32 (m, 5H), 7.10 (s, 1H), 5.18 (d, J = 15.1 Hz, 4H), 4.85 (t, J = 6.4 Hz, 2H), 3.99 (s, 3H), 3.95 (s, 3H), 3.88 (s, 3H), 3.19 (t, J = 6.4 Hz, 2H). 13 C-NMR (126 MHz) δ 168.7, 151.4, 148.8, 148.6, 145.6, 141.3, 137.6, 135.3, 132.9, 128.5, 128.3 (2), 128.1 (2), 126.6, 123.2, 121.0, 119.6, 118.7, 111.1, 108.5, 69.2, 66.1, 57.0, 56.0, 55.7, 55.3, 25.9. HRMS: calcd. for C29 H28 ClNO6 [M]+ : 486.1911, found: 486.1916. 2,3,10-Trimethoxy-9-p-tolylformylmethylenoxy protoberberine chloride (4d). Compound 3 (500 mg, 1.34 mmol) was treated with 2-bromo-40 -methyl acetophenone (571.0 mg, 2.68 mmol) according to the general procedure to give the desired product 4d. Yield: 21%; yellow solid; m.p. 228–230 ◦ C; 1 H-NMR (500 MHz) δ 10.02 (s, 1H), 9.05 (s, 1H), 8.18 (d, J = 10.0 Hz, 1H), 8.01 (d, J = 10.0 Hz, 1H), 7.90 (d, J = 10.0 Hz, 2H), 7.74 (s, 1H), 7.39 (d, J = 10.0 Hz, 2H), 7.11 (s, 1H), 5.87 (s, 2H), 4.97 (t, J = 5.0 Hz, 2H), 3.95 (d, J = 5.0 Hz, 6H), 3.88 (s, 3H), 3.25 (t, J = 5.0 Hz, 2H), 2.40 (s, 3H). 13 C-NMR (126 MHz) δ 194.5, 151.9, 149.3, 149.2, 146.4, 144.9, 142.5, 138.2, 133.6, 132.0, 130.0 (2), 129.1, 128.3 (2), 127.2, 123.3, 121.5, 120.2, 119.4, 111.7, 109.2, 75.2, 57.6, 56.6, 56.3, 56.0, 26.5, 21.7. HRMS: calcd. for C29 H28 ClNO5 [M]+ : 470.1962, found: 470.1961. 2,3,10-Trimethoxy-9-m-methoxyphenylformylmethylenoxy protoberberine chloride (4e). Compound 3 (500 mg, 1.34 mmol) was treated with 2-bromo-30 -methoxyacetophenone (613.9 mg, 2.68 mmol) according to the general procedure to give the desired product 4e. Yield: 20%; red brown solid; m.p. 200–202 ◦ C; 1 H-NMR (400 MHz) δ 10.02 (s, 1H), 9.07 (s, 1H), 8.18 (d, J = 10.0 Hz, 1H), 8.02 (d, J = 10.0 Hz, 1H), 7.74 (s, 1H), 7.60 (d, J = 10.0 Hz, 1H), 7.52–7.47 (m, 2H), 7.30–7.27 (m, 1H), 7.12 (s, 1H), 5.89 (s, 2H), 4.98 (t,

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J = 5.0 Hz, 2H), 3.95 (s, 6H), 3.88 (s, 3H), 3.83 (s, 3H), 3.24 (t, J = 5.0 Hz, 2H). 13 C-NMR (101 MHz) δ 194.2, 159.4, 151.4, 148.8, 148.6, 145.7, 141.9, 137.6, 135.3, 133.0, 130.1, 128.5, 126.7, 122.8, 121.0, 120.1, 119.8, 119.7, 118.8, 112.2, 111.2, 108.7, 74.8, 57.0, 56.1, 55.8, 55.5, 55.3, 26.0. HRMS: calcd. for C29 H28 ClNO6 [M]+ : 486.1911, found: 486.1916. 2,3,10-Trimethoxy-9-p-trifloromethylphenylformylmethylenoxy protoberberine chloride (4f). Compound 3 (373 mg, 1 mmol) was treated with 4-(trifluoromethyl) phenacyl bromide (534 mg, 2 mmol) according to the general procedure to give the desired product 4f. Yield: 29%; red brown solid; m.p. 240–242 ◦ C; 1 H-NMR (400 MHz) δ 10.01 (s, 1H), 9.05 (s, 1H), 8.21–8.18 (m, 3H), 8.03–7.96 (m, 3H), 7.74 (s, 1H), 7.12 (s, 1H), 5.93 (s, 2H), 4.97 (t, J = 5.0 Hz, 2H), 3.95 (d, J = 5.0 Hz, 6H), 3.88 (s, 3H), 3.25 (t, J = 5.0 Hz, 2H). 13 C-NMR (101 MHz) δ 194.0, 151.4, 148.8, 148.6, 145.7, 141.7, 137.7, 137.3, 133.0, 128.6 (2), 128.5, 126.7, 125.8 (2), 125.3, 124.9, 122.94, 120.9, 119.77, 118.7, 111.2, 108.7, 75.0, 57.0, 56.1, 55.8, 55.5, 25.9. HRMS: calcd. for C29 H25 ClF3 NO5 [M]+ : 524.1679, found: 524.1686. 2,3,10-Trimethoxy-9-benzyloxyformyloxy protoberberine chloride (4g). Compound 3 (373 mg, 1 mmol) was treated with benzyl chloroformate (682.4 mg, 4 mmol) according to the general procedure to give the desired product 4g. Yield: 21%; yellow solid; m.p. 98–100 ◦ C; 1 H-NMR (400 MHz) δ 10.02 (s, 1H), 9.16 (s, 1H), 8.29 (dd, J = 10.0 Hz, J = 25.0 Hz, 2H), 7.74 (s, 1H), 7.53–7.43 (m, 5H), 7.11 (s, 1H), 5.41 (s, 2H), 4.96 (t, J = 5.0 Hz, 2H), 4.03 (s, 3H), 3.95 (s, 3H), 3.88 (s, 3H), 3.24 (t, J = 5.0 Hz, 2H). 13 C-NMR (101 MHz) δ 151.6, 150.0, 148.6, 144.2, 138.4, 134.6, 133.3, 132.8, 128.7, 128.6, 128.5 (2), 128.1 (2), 126.9, 125.9, 120.4, 120.2, 118.7, 111.2, 108.7, 70.7, 57.2, 56.1, 55.8, 55.4, 45.3, 25.7. HRMS: calcd. for C28 H26 ClNO6 [M]+ : 472.1755, found: 472.1758. 3.2.3. General Procedure for the Synthesis of Compounds 5a–g To a stirred solution of JTH (100 mg, 0.29 mmol) in anhydrous CH3 CN or DMF, K2 CO3 (122 mg, 0.88 mmol) was added and heated to 70 ◦ C. Then R1 Br or benzyl chloroformate (2–4 eq) was added and stirred for 5–6 h. The mixture was cooled to precipitate completely, filtrated and washed by CH2 Cl2 to afford compounds 5a–g. 2,9,10-Trimethoxy-3-ethyloxyformylmethylenoxy protoberberine chloride (5a). JTH (373 mg, 1 mmol) was treated with ethyl bromoacetate (668 mg, 4 mmol) according to the general procedure to give the desired product 5a. Yield: 35%; yellow solid; m.p. 234–236 ◦ C; 1 H-NMR (500 MHz) δ 9.91 (s, 1H), 9.07 (s, 1H), 8.23 (d, J = 10.0 Hz, 1H), 8.06–8.04 (m, 1H), 7.76 (s, 1H), 7.04 (s, 1H), 4.94 (t, J = 5.0 Hz, 2H), 4.92 (s, 2H), 4.20 (dd, J = 5.0 Hz, J = 15.0 Hz, 2H), 4.10 (s, 3H), 4.08 (s, 3H), 3.97 (s, 3H), 3.19 (t, J = 5.0 Hz, 2H), 1.24 (t, J = 5.0 Hz, 3H). 13 C-NMR (126 MHz) δ 168.2, 150.2, 149.5, 148.6, 145.4, 143.5, 137.4, 132.9, 128.2, 126.6, 123.3, 121.3, 112.0, 119.7, 112.4, 109.1, 64.9, 61.8, 60.7, 56.9, 56.1, 55.2, 25.7, 14.0. HRMS: calcd. for C24 H26 ClNO6 [M]+ : 424.1755, found: 424.1751. 2,9,10-Trimethoxy-3-pivalylmethylenoxy protoberberine chloride (5b). JTH (373 mg, 1 mmol) was treated with 1-bromo-3,3-dimethyl-2-butanone (358.1 mg, 2 mmol) according to the general procedure to give the desired product 5b. Yield: 28%; yellow solid; m.p. 223–225 ◦ C; 1 H-NMR (500 MHz) δ 9.90 (s, 1H), 9.05 (s, 1H), 8.23 (d, J = 5.0 Hz, 1H), 8.05 (d, J = 5.0 Hz, 1H), 7.74 (s, 1H), 6.88 (s, 1H), 5.27 (s, 2H), 4.94 (t, J = 5.0 Hz, 2H), 4.10 (s, 3H), 4.08 (s, 3H), 3.96 (s, 3H), 3.18 (t, J = 5.0 Hz, 2H), 1.20 (s, 9H). 13 C NMR (126 MHz) δ 209.2, 150.2, 145.0, 148.6, 145.3, 143.5, 137.5, 132.9, 128.1, 126.6, 123.3, 121.2, 119.8, 119.1, 112.0, 109.1, 68.8, 61.8, 56.9, 56.1, 55.2, 42.4, 25.8, 25.6 (3). 13 C-NMR (126 MHz) δ 209.2, 150.2, 145.0, 148.6, 145.3, 143.5, 137.5, 132.9, 128.1, 126.6, 123.3, 121.2, 119.8, 119.1, 112.0, 109.1, 68.8, 61.8, 56.9, 56.1, 55.2, 42.4, 25.8, 25.6 (3). HRMS: calcd. for C26 H30 ClNO5 [M]+ : 436.2119, found: 436.2118. 2,9,10-Trimethoxy-3-benzyloxyformylmethylenoxy protoberberine chloride (5c). JTH (373 mg, 1 mmol) was treated with benzyl bromoacetate (916.2 mg, 4 mmol) according to the general procedure to give the desired product 5c. Yield: 38%; yellow solid; m.p. 217–219 ◦ C; 1 H-NMR (500 MHz) δ 9.90 (s, 1H), 9.06 (s, 1H), 8.23 (d, J = 10.0 Hz, 1H), 8.04 (d, J = 10.0 Hz, 1H), 7.75 (s, 1H), 7.40–7.35 (m, 4H), 7.03 (s, 1H), 5.23 (s, 2H), 5.01 (s, 2H), 4.93 (t, J = 5.0 Hz, 2H), 4.10 (s, 3H), 4.08 (s, 3H), 3.96 (s, 3H), 3.16 (t, J = 5.0 Hz,

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2H). 13 C-NMR (126 MHz) δ 168.2, 150.2, 149.5, 148.6, 145.4, 143.5, 137.3, 135.5, 132.9, 128.34 (2), 128.1, 128.1, 127.94 (2), 126.6, 123.3, 121.3, 120.0, 119.73, 112.4, 109.2, 66.0, 64.9, 61.8, 56.9, 56.1, 55.2, 25.8. HRMS: calcd. for C29 H28 ClNO6 [M]+ : 486.1911, found: 486.1910. 2,9,10-Trimethoxy-3-p-methylphenylformylmethylenoxy protoberberine chloride (5d). JTH (373 mg, 1 mmol) was treated with 2-bromo-40 -methylacetophenone (426.1 mg, 2 mmol) according to the general procedure to give the desired product 5d. Yield: 38%; yellow solid; m.p. 218–220 ◦ C; 1 H-NMR (500 MHz) δ 9.89 (s, 1H), 9.06 (s, 1H), 8.23 (d, J = 10.0 Hz, 1H), 8.05 (d, J = 10.0 Hz, 1H), 7.76 (s, 1H), 7.65–7.63 (m, 1H), 7.54–7.51 (m, 2H), 7.32–7.29 (m, 1H), 7.06 (s, 1H), 5.75 (s, 2H), 4.93 (t, J = 5.0 Hz, 2H), 4.10 (s, 3H), 4.08 (s, 3H), 3.99 (s, 3H), 3.85 (s, 3H), 3.16 (t, J = 5.0 Hz, 2H). 13 C-NMR (126 MHz) δ 193.5, 159.3, 150.2, 150.0, 148.6, 145.3, 143.5, 137.5, 135.4, 132.9, 130.0, 128.2, 126.6, 123.3, 121.3, 120.2, 119.8, 119.6, 119.2, 112.5, 112.3, 109.1, 70.5, 61.8, 56.9, 56.1, 55.4, 55.2, 25.7. HRMS: calcd. for C29 H28 ClNO5 [M]+ : 470.1962, found: 470.1965. 2,9,10-Trimethoxy-3-m-methoxyphenylformylmethylenoxy protoberberine chloride (5e). JTH (373 mg, 1 mmol) was treated with 2-bromo-30 -methoxyacetophenone (458.1 mg, 2 mmol) according to the general procedure to give the desired product 5e. Yield: 36%;yellow solid; m.p. 218–220 ◦ C; 1 H-NMR (500 MHz) δ 9.89 (s, 1H), 9.06 (s, 1H), 8.23 (d, J = 10.0 Hz, 1H), 8.05 (d, J = 10.0 Hz, 1H), 7.96–7.94 (m, 2H), 7.76 (s, 1H), 7.42–7.40 (m, 2H), 7.04 (s, 1H), 5.71 (s, 2H), 4.93 (t, J = 5.0 Hz, 2H), 4.10 (s, 3H), 4.08 (s, 3H), 3.99 (s, 3H), 3.15 (t, J = 5.0 Hz, 2H), 2.42 (s, 3H). 13 C-NMR (126 MHz) δ 193.2, 150.2, 150.1, 148.6, 145.3, 144.3, 143.5, 137.5, 132.9, 131.6, 129.3 (2), 128.2, 127.9 (2), 126.6, 123.3, 121.3, 119.8, 119.2, 112.3, 109.1, 70.3, 61.8, 56.9, 56.1, 55.2, 25.7, 21.2. HRMS: calcd. for C29 H28 ClNO6 [M]+ : 486.1911, found: 486.1914. 2,9,10-Trimethoxy-3-p-trifloromethylphenylformylmethylenoxy protober berine chloride (5f). JTH (373 mg, 1 mmol) was treated with 4-(trifluoromethyl) phenacyl bromide (534.1 mg, 2 mmol) according to the general procedure to give the desired product 5f. Yield: 35%; yellow solid; m.p. 213–215 ◦ C; 1 H-NMR (500 MHz) δ 9.90 (s, 1H), 9.07 (s, 1H), 8.25–8.22 (m, 3H), 8.05 (d, J = 10.0 Hz, 1H), 8.01–7.99 (m, 2H), 7.77 (s, 1H), 7.13 (s, 1H), 5.81 (s, 2H), 4.93 (t, J = 5.0 Hz, 2H), 4.10 (s, 3H), 4.08 (s, 3H), 3.99 (s, 3H), 3.16 (t, J = 5.0 Hz, 2H). 13 C-NMR (126 MHz) δ 193.3, 150.2, 149.9, 148.6, 145.4, 143.5, 137.5, 137.3, 132.9, 132.8, 128.7, 128.2, 126.6, 125.7 (2), 124.7, 123.3, 122.5, 121.3, 119.9, 119.4, 112.4, 109.1, 70.7, 61.8, 56.9, 56.1, 55.2, 25.7. HRMS: calcd. for C29 H25 ClF3 NO5 [M]+ : 524.1679, found: 524.1679. 2,9,10-Trimethoxy-3-benzyloxyformyloxy protoberberine chloride (5g). JTH (373 mg, 1 mmol) was treated with benzyl chloroformate (682.4 mg, 4 mmol) according to the general procedure to give the desired product 5g. Yield: 36%; yellow solid; m.p. 140–142 ◦ C; 1 H-NMR (500 MHz) δ 9.99 (s, 1H), 9.22 (s, 1H), 8.27 (d, J = 10.0 Hz, 1H), 8.10 (d, J = 10.0 Hz, 1H), 7.95 (s, 1H), 7.46–7.41 (m, 6H), 5.32 (s, 2H), 4.99 (t, J = 5.0 Hz, 2H), 4.12 (s, 3H), 4.09 (s, 3H), 3.97 (s, 3H), 3.24 (t, J = 5.0 Hz, 2H). 13 C-NMR (126 MHz) δ152.1, 150.8, 150.7, 145.9, 143.7, 141.3, 136.5, 134.8, 132.5, 128.5, 128.5(2), 128.2(2), 127.9, 126.6, 125.7, 123.6, 122.3, 121.7, 121.4, 110.1, 70.0, 61.9, 56.9, 56.5, 55.2, 25.3. HRMS: calcd. for C28 H26 ClNO6 [M]+ : 472.1755, found: 472.1753. 3.3. Biology Assays 3.3.1. CPE Inhibition Assay for Anti-EV71 The anti-EV71 activities of all tested compounds were detected by the virus-induced CPE assay. Briefly, cells (3 × 104 cells/well) were plated into 96-well culture plates and incubated for 16 h. Then, remove the medium and infected cells with EV71 of 100 × TCID50 (50% tissue culture infective doses) in serum-free medium for 1 h at 37 ◦ C. After that, the unbound viruses were removed and various concentrations of tested compounds were supplemented for incubation of another 72 h. IC50 defined as the minimal concentration required to inhibit 50% of CPE was determined by Reed & Muench method in 48 h. TC50 defined as the concentration that leads to the 50% of CPE which determined by

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the same method in 72 h. In addition, the cells were stained the cells were colored with 0.5% crystal violet in 20% ethanol for 15 min at room temperature and the cells were imaged after rinsed with PBS. 3.3.2. Cytotoxicity Assay Cytotoxicity of all target compounds in Vero cells was analyzed by CCK (TransGen Biotech, Beijing, China) assay. In brief, cells in exponential phase (3 × 104 cells/well) were seeded into 96-well culture plates and incubated overnight. Then, the medium was removed and different concentrations of all target compounds were applied in duple. After 48 h incubation, the cytotoxicity of all target compounds was determined by CCK assay. The signals were read at 450 nm on Enspire (Perkin Elmer, Waltham, MA, USA). The TC50 was defined as the concentration that inhibits 50% cellular growth in comparison with the controls. It is calculated by the Reed and Muench method. 3.3.3. Western Blot Analysis Cells were lysed in the M-PER mammalian protein extraction reagent (Thermo, Rockford, IL, USA) containing halt protease inhibitor single-use cocktail (Thermo). The protein concentration was determined by BCA Protein Assay Kit (Thermo). Equal amount of samples (15 µg proteins) were denatured and applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoresis products were transferred to a polyvinylidenefluoride (PVDF) film and blocked by 5% (w/v) milk or BSA at room temperature. An hour later, PVDF membranes were incubated at room temperature with specific primary antibody. After a standard washing, membranes were incubated with horse radish peroxidase (HRP)-labeled secondary antibody. The signals were detected using ECL detection kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The primary antibodies used in the experiment included β-actin, p-p44/p42 MAPK, p44/p42 MAPK, p-MEK, MEK, p-JNK, JNK, p-AKT, AKT, PI3KIII, LC3B, Beclin-1 (Cell Signaling Technology, Danvers, MA, USA), and EV71-VP1 (Abnova, Taibei, China). HRP-labeled secondary antibodies included goat anti-rabbit and anti-mouse HRP-labeled antibodies (Cell Signaling Technology). 3.3.4. qRT-PCR Quantification Vero cells (9 × 105 cells/well) were plated into 6-well culture plates and incubated for 16 h. The medium was removed and cells were infected with EV71 for 1 h (H). Various concentrations of compound 2d and BBR were supplemented for incubation of another 24 h. Total RNA of the infected cells was isolated using the RNeasy Mini kit (QIAGEN, Hilden, Germany) and the information of the primer was listed in the Table 2. Table 2. Oligonucleotides used for real-time RT-PCR. Oligonucleotide

Sequence (50 –30 )

50 VP1(EV71) 30 VP1(EV71) 50 β-actin 30 β-actin

50 -GCAGCCCAAAAGAACTTCAC-30 50 -ATTTCAGCAGCTTGGAGTGC-30 0 5 -TGACGGGGTCACCCACACTGTGCCCATCTA-30 50 -CTAGAAGCATTTGCGGTGGACGATG-30

One-step qRT-PCR was performed with SuperScript III Platinum SYBR Green One-step RT-PCR Kit (Invitrogen, Carlsbad, CA, USA) using the ABI 7500 Fast real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). PCR assay was carried out in a 25 µL volume and the target fragment amplification was carried out as follows: reverse transcription at 50 ◦ C for 3 min; initial activation of HotStar Taq DNA Polymerase at 95 ◦ C for 10 min; 40 cycles in two steps: 95 ◦ C for 15 s, 60 ◦ C for 30 s.

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3.3.5. Statistical Analysis Data were expressed as the mean ± standard error of the mean and analyzed with one-way ANOVA using MTLAB software (8.6, MathWorks, 2015, Natick, MA, USA). A threshold of p < 0.05 was defined as statistically significant. 4. Conclusions Taking BBR as the lead, 23 new BBR derivatives were synthesized and examined for their anti-EV71 activities against different genotype strains with CPE assay. SAR indicated that introduction of a suitable 9-ether group might be beneficial for potency. Among them, compound 2d exhibited most potent activities against all tested EV71 strains with IC50 values in the range of 7.12–14.8 µM. Its effect was further confirmed in a dose-dependent manner both in RNA and protein level, better than that of BBR. The preliminary mechanism revealed that compound 2d could inhibit the activation of MEK/ERK signaling pathway. Furthermore, 2d could suppress the EV71-induced autophagy by activating AKT and inhibiting the phosphorylation of JNK and PI3KIII proteins. Compound 2d owned a potent anti-EV71 effect with new mechanism of action, has been selected for next investigation. We consider BBR derivatives to be a new class of antiviral agents against EV71 through targeting host components. The results provided the powerful information for further development of this kind of compounds into a novel family of antiviral candidates against EV71, with an advantage of broad-spectrum anti-EV71 potency. Supplementary Materials: The following are available online. Figure S1: 1 H-NMR, 13 C-NMR, HRMS-ESI spectra. Author Contributions: Y.-X.W. performed part of synthetic experiments and wrote the paper, L.Y. and H.-Q.W. performed the biological assay, X.-Q.Z. and T.L. was responsible for literature search, Y.-H.L. and Q.-X.Z. conceived and designed the chemistry experiments, Y.-H.L. conceived and designed the biology experiments, D.-Q.S. designed the target compounds and chemistry experiments. Funding: This work was supported by the CAMS initiative for innovative medicine 2016-12M-1-011 and the National S&T Major Special Project on Major New Drug Innovation (2018ZX09711003-005-004) and the National Natural Science Foundation of China (81321004, 81473248 and 81773782). Acknowledgments: The authors thank center for analysis and testing of Institute of Materia Medica and Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences for their contributions to the determination of HR-MS, 1 H-NMR and 13 C-NMR. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 2a–i, 4a–g and 5a–g are available from the authors. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).