Chinese Journal of Natural Medicines  2018, Vol. 16Issue (10): 774-781  DOI: 10.1016/S1875-5364(18)30117-1
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Cite this article as: 

YAO Ling, WU Ling-Ling, LI Qian, HU Qin-Mei, ZHANG Shu-Yuan, LIU Kang, JIANG Jian-Qin. Novel berberine derivatives: Design, synthesis, antimicrobial effects, and molecular docking studies[J]. Chinese Journal of Natural Medicines, 2018, 16(10): 774-781.
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Research funding

This work was supported by the Graduate Students Scientific Research Innovation Projects of Jiangsu Province (No. CXZZ11-0804) and Students Training Innovation Program of China Pharmaceutical University (201810316155)

Corresponding author

Tel: 13912979427, E-mail: liukangcpu@aliyun.com (LIU Kang)
Tel: 13913982651, E-mail: njjjq@aliyun.com (JIANG Jian-Qin)

Article history

Received on: 28-May-2018
Available online: 20 October, 2018
Novel berberine derivatives: Design, synthesis, antimicrobial effects, and molecular docking studies
YAO Ling , WU Ling-Ling , LI Qian , HU Qin-Mei , ZHANG Shu-Yuan , LIU Kang , JIANG Jian-Qin     
School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China
[Abstract]: A series of berberine derivatives were synthesized by introducing substituted benzyl groups at C-9. All these synthesized compounds (4a-4m) were screened for their in vitro antibacterial activity against four Gram-positive bacteria and four Gram-negative bacteria and evaluated for their antifungal activity against three pathogenic fungal strains. All these compounds displayed good antibacterial and antifungal activities, compared to reference drugs including Ciprofloxacin and Fluconazole; Compounds 4f, 4g, and 4l showed the highest antibacterial and antifungal activities. Moreover, all the synthesized compounds were docked into topoisomerase Ⅱ-DNA complex, which is a crucial drug target for the treatment of microbial infections. Docking results showed that H-bond, π-π stacked, π-cationic, and π-anionic interactions were responsible for the strong binding of the compounds with the target protein-DNA complex.
[Key words]: Antimicrobial drugs     Berberine derivatives     Molecular docking     Topoisomerase Ⅱ DNA gyrase     SAR    
Introduction

Although the focus of health care may be shifted from infectious to non-communicable diseases [1], combating infectious diseases is still a global challenge in the 21st century, particularly in the developing world, due to the poor economy and ever-increasing resistance of pathogens against common therapeutic agents [2-5]. According to the Global Burden of Disease Study 2013, about 17% of all deaths (≈ 9.2 million deaths) are related to infectious diseases, which are mainly caused by the pathogenic microorganisms such as bacteria, viruses, fungi, and parasites [1, 4]. In addition to the high mortality, infectious diseases also constitute a financial burden to healthcare systems all over the world [6-7]. Therefore, the development of effective and affordable small-molecule antimicrobial drugs is urgently needed. With genomics and associated new technologies, it is believed that a plethora of new drugs lacking cross-resistance to clinically used antimicrobial drugs would be developed [8-10]. Unfortunately, the efforts have not produced much outcome. Thus, continued researches are still dedicated to filling the unmet antimicrobial medications [11-12].

Berberine (BBR) is an isoquinoline alkaloid occurring in numerous medicinal plants of Coptis Salisb. and Phellodendron, initially wellknown for the significant effects on gastroenteritis and bacillary dysentery in Chinese and Ayurvedic medicinal systems [13-15]. Over the years, many other pharmacological effects of BBR, such as antimicrobial, hepatoprotective [16], anti-hyperlipidemic [17], anticancer [18], anti-diabetic [19], anti-inflammatory, and anti-arrhythmic activities, have been reported [13]. Especially, BBR displays therapeutic efficacy against methicilin resistant Staphylococcus aureus [20], multidrug resistant enterovirulent Escherichia coli [21], and H1N1 influenza A virus [22]. Although BBR shows capacity in the challenging microbial infection treatment, its antimicrobial activity in vivo is not effective enough to replace existing antimicrobial agents in the clinic [23-24]. Therefore, it is of significance to improve antimicrobial activity of BBR by a variety of strategies. One of the most significant strategies is using BBR as a leading compound to modify its structure, thus to obtain new synthetic antimicrobial drugs [25-26].

Park et al. have synthesized a series of 13-(substituted benzyl) BBR and berberrubine derivatives, which showpotent antifungal activity against Candida species [27]. Siritron et al. have synthesized a series of 13-substituted berberine NorA inhibitor hybrid and 13-arylmethyl ether hybrid structures, one of which shows better antibacterial activity than BBR (MIC Staphylococcus aureus, 1.7 µmol·L-1) [28]. Li et al. have reported the synthesis and structure-activity relationship of 8-substituted protoberberine derivatives as a novel class of antitubercular agents [29]. Lo et al. have found that the lipophilic substitute of 9-O-alkyl-and 9-O-terpenyl BBR derivatives play an important role in the anti-cancer activity [30]. Zhang et al. have designed and synthesized a series of novel 9-O-(lipophilic group substituted) BBR derivatives displaying dramatically increased hypoglycemic activity and lower cytotoxicity compared with BBR [31]. These reports have suggested the importance of introduction of lipophilic group and aromatic group, particularly, emphasizing the fact that CF3-, NO2-, CN- and halogens- substituted molecules play a significant role in medicinal chemistry. Based on these researches, we intended to enhance antimicrobial activity of BBR by structural modification with lipophilic aromatic groups in the present study. A series of 9-O-(substituted benzyl) BBR derivatives (compounds 4a-4m) were designed and synthesized. Their antimicrobial activities were evaluated in vitro, and preliminary structure-activity relationships were also explored. Additionally, molecular docking was carried out to validate the proposed SAR.

Results and Discussion Compound design and Chemistry

As illustrated in Scheme 1, all the newly designed compounds were synthesized based on berberrubine, which was an active metabolite of BBR after first pass metabolism with enhanced water solubility, but having poorer biological activity, compared with BBR owing to the hydroxyl group at C-9. In these derivatives, substituted benzyl moieties were added through-O-bond at C-9, meanwhile four key antimicrobial pharmacophores of BBR were maintained: (Ⅰ) the methylenedioxy, (Ⅱ) the cationic nitrogen, (Ⅲ) methoxyl group at C-10, and (Ⅳ) the bis-isoquinoline core [32].

Scheme 1 Design and key antimicrobial blocks of 9-O-(substituted benzyl) BBR derivatives

The synthetic routes of 9-O-(substituted benzyl) BBR derivatives (compounds 4a-4m) are outlined in Scheme 2. Briefly, berberine (1) was mixed with pyridine hydrochloride and butyric anhydride at 135 ℃ to get Compound 2 and the progress of the reaction was monitored with TLC. Followed by filtrating, washing and drying, 10% NaOH hydrolysis was conducted to gain berberrubine 3. Then, berberrubine was condensed with substituted benzyl bromide to afford target compounds 4a-4m. Their structures were characterized by 1H NMR spectroscopy, 13C NMR spectroscopy, and MS spectroscopy.

Scheme 2 Synthesis of 9-O-(substituted benzyl) BBR derivatives. Reagents and conditions: (a) pyridine hydrochloride, butyric anhydride, 135 ℃; (b) 10% NaOH, rt, conc. HCl; (c) acetonitrile, K2CO3, KI, reflux
Antibacterial activities

The bacterial strains used in the study were as follows: (1) Escherichia coli (CICC 10389), (2) Pseudomonas aeruginosa (CICC 10419), (3) Clostridium perfringens (CICC 22949), (4) Staphylococcus aureus (CICC 23656), (5) Salmonella enteritidis (CICC 24119), (6) Shigella dysenteriae (CICC 23829), (7) Streptococcus pyogenes (CICC 10356), and (8) S. aureus (MRSA, ATCC 700699). As shown in Table 1, all the newly synthesized scaffolds showed moderate antibacterial activities, among which Compounds 4f, 4g, and 4l in particular, displayed a broader antibacterial spectrum with much lower MICs, compared with BBR. Although all the molecules displayed comparable or even weaker inhibitory effects on most of tested bacterium compounds compared with the standard drug Ciprofloxacin (CIP), it was particularly noteworthy that Compounds 4f, 4g, and 4l showed superior activities than the standard drug in assays against resistant strains (S. aureus, MRSA).

Table 1 Antibacterial activities of BBR and compounds 4a-4m
Antifungal activities

We next evaluated their effects on various fungal strains, including the following: (1). Candida albicans (CICC 32819), (2). Cryptococcus neoformans (ATCC 76484), and (3). Candida parapsilosis (CICC 1973). All these compounds showed good inhibition activity against all the tested pathogenic fungal strains. Interestingly, there were some parallels between the antifungal and antibacterial assay results. Compounds 4f, 4g, and 4l also showed the highest antifungal activity (4f being one of the most potent with MIC 1.65 µmol·L-1 against C. albicans, MIC 3.29 µmol·L-1 against C. neoformans, MIC 6.59 µmol·L-1 against C. albicans), while the other compounds which were less potent in antibacterial assays, remained so against fungal strains. The results are shown in Table 2.

Table 2 Antifungal activities of BBR and compounds 4a-4m
Structure-activity relationship (SAR)

In accordance with literatures, the bis-isoquinoline core, the cationic nitrogen, the methylenedioxy and methoxyl group at C-10 are key blocks for the antimicrobial activities.

For the structure-activity relationship study of benzyl moiety, we firstly introduced electron-donating groups to benzyl moiety. Compared to compound 4a, methyl substitution at meta- or ortho-position of benzyl (4h and 4m) and methoxy substitution at meta- or para- (4i and 4j) showed comparable activities, while the 4-t-Bu substituted compound led to enhanced antimicrobial activities.

We next explored the inductive effects by introducing the electron-withdrawing groups. All the compounds having an electron withdrawing group in benzyl moiety showed enhanced antibacterial activity against all tested microorganisms. Especially, CF3-, NO2-, CN- substituted compounds (4f, 4g, and 4l) showed good antibacterial activities against S.aureus, C. perfringens and Gram-negative microorganisms such as S. dysenteriae. The benzene ring containing the 4-NO2 and 4-CF3 showed excellent antifungal activity.

All the SAR studies revealed that various substituents like electron donating and electron withdrawing groups in benzyl moiety were essential for the antimicrobial activities, so we formulated a hypothesis that the substituted benzyl moiety is one of the important activity binding centers with potential targets like type Ⅱ topoisomerase.

Molecular docking studies

DNA gyrase is a kind of unique type Ⅱ topoisomerase which is responsible for the super-coiling activity of DNA in bacteria. And our leading compound BBR has been proven to be a potential topoisomerase Ⅱ Inhibitor [33-34]. To rationalize the proposed SAR and the interactions between our compounds and DNA gyrase, the docking studies were carried out using DS 3.0. Table 3 summarizes the docking scores and interactions of BBR derivatives with DNA gyrase. All the synthesized compounds showed good binding interactions with the receptor and showed LibDock score values in a range from 131.081 to 158.252. The most potent compound 4f showed a maximum dock score value of 158.252. All the docking results obtained were compared with the antimicrobial test results. As shown in Fig. 1, we found the changing trend of [1/score] was consistent with antimicrobial test results, which further verified our aforementioned hypothesis.

Table 3 Docking scores of BBR and compounds 4a-4m
Figure 1 The relationship between antimicrobial activities of compounds and docking score

In order to get further insights into the structural basis for its activity, compound 4f was analyzed in more details. As shown in Fig. 2, the highly aromatic bis-isoquinoline core of 4f sat between two stretched DNA base pairs (DG E: 10, DG F: 10, DC E: 11 and DC F: 11) through π-π stacked and π-cationic interactions. The CF3- substituted benzyl moiety resided in an active pocket at the interface of the two GyrA subunits, ultimately leading to a suboptimal positioning of a (charged) H-bond acceptor that could interact with MET B: 1121 and ARG B: 1122. Furthermore, the benzyl moiety interacted with the ASP B: 1083 through a hydrogen bond and π-anionic interaction. Meanwhile, the hydrophobic moiety of 4f established alkyl interactions and van der Waals interactions with the surrounding hydrophobic residues and DNA base pairs.

Figure 2 Predicted binding mode for compound 4f with DNA gyrase and DNA complex (PDB code: 2XCS). (A) Top view of compound 4f (yellow sticks) bound to the charge surface of protein-DNA complex. (B) 3D ligand interaction diagram of compound 4f (yellow sticks) bound to key protein residues and nucleotide. (C) 2D ligand interaction diagram of docking model of compound 4f
Experimental Chemistry General experimental procedures

All the chemicals, reagents, and solvents used in the present study were purchased from commercial companies (Aladdin reagent (Shanghai) Co., Ltd., Shanghai, China). Solvents used for HPLC analysis were HPLC grade. When necessary, the solvents were used with further purification and dryness. Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 precoated plates (Qingdao Ocean Chemical Industry Co., Qingdao, Ltd., China), and spots were visualized by ultraviolet light at 365 nm. Column chromatography was performed on silica gel (200-300 mesh, Qingdao Ocean Chemical Industry Co., Qingdao, Ltd., China) for the purification of intermediates and final compounds. Melting points were determined using a Mel-TEMP Ⅱ melting point apparatus (Cole-Parmer Instrument (Shanghai) Co., Ltd., Shanghai, China). 1H NMR and 13C NMR spectra of compounds were recorded on a Bruker Avance 300 spectrometer (Bruker Corporation, Germany), when dissolved in DMSO-d6 with 0.03% TMS as an internal standard, unless otherwise specified. Chemical shifts (δ) were reported in ppm with reference to solvent signal. Coupling constants (J) were given in hertz and peak multiplicities were reported as singlet (s), doublet (d), triplet (t), multiplet (m). Mass spectrometry (MS) was performed on a ESI time-of-flight spectrometer. Purities of all final compounds were determined by analytical HPLC on an Shimadzu SCL-10A system (Shimadzu Corporation, Japan) with a HEDERA ODS-2 column (5 µm, 250 mm × 4.6 mm i.d., Jiangsu Hanbon Science & Technology Co., Ltd., China) detected at 245 nm and 345 nm. The gradient elution program was conducted with Solvent A (water containing 0.1% formic acid and 2 mmol·L-1 ammonium acetate) and Solvent B (acetonitrile) as follows: 10%-90% B over 40 min, 90%-10% B over 5 min, and 10% B over 5 min at a flow rate of 1.0 mL·min-1 and the column temperature of 25 ℃. The purities of all the biologically evaluated compounds were determined to be ≥ 90%.

General procedure of Synthesis and spectral data General procedure for synthesis of compound 2

To a solution of Butyric anhydride (120 mL), the starting compound berberine 1 (10 g, 28.1 mmol) and pyridine hydrochloride (15 g, 129.8 mmol) were added and stirred at 135 ℃ for 8-9 h. The mixture was cooled at ice bath while stirring for 3 h. The precipitate was collected by filtration, washed with cold water and dried over an infrared lamp to obtain compound 2 as a yellow solid and used in the next step without further purification, yield 89.2% (10.68 g, 24.6 mmol).

General procedure for synthesis of compound 3

To a solution of 10% NaOH (100 mL), compound 2 was added and stirred at room temperature for 2 h. The mixture was acidified with conc. HCl until pH 2-3. Then, the precipitate was collected by filtration and washed with cold water and diethyl ether. The crude product was purified by recrystallization and dried over an infrared lamp to give the refined compound 3 as a vermeil solid, yield 90.5% (8.38 g, 23.2 mmol).

General procedure for synthesis of compounds 4a-4m

To a mixture solution of compound 3 (0.2 g, 0.54 mmol), K2CO3 (0.22 g, 1.6 mmol), and KI (0.11g, 1.1 mmol) in anhydrous acetonitrile (20 mL), substituted benzyl bromide compounds were added to prepare final compounds. The mixture was stirred for 3-5 h at 70-75 ℃ and the solution was evaporated. KI and K2CO3 in the resulting residue was dissolved by the addition of water. The precipitate was collected by filtration and washed with cold water and diethyl ether to get the crude product. Then, the crude product was chromatographed on silica gel (CH3Cl/MeOH/Et3N = 50 : 1 : 0.6) to afford compounds 4a-4m.

Compound 4a. Yellow powder, yield: 62.3%, mp 233-235 ℃. 1H NMR (400 MHz, DMSO-d6) δ 9.75 (s, 1H), 8.95 (s, 1H), 8.22 (d, J = 9.1 Hz, 1H), 8.01 (d, J = 9.1 Hz, 1H), 7.80 (s, 1H), 7.60 (d, J = 7.1 Hz, 2H), 7.39 (dt, J = 13.7, 6.9 Hz, 3H), 7.09 (s, 1H), 6.18 (s, 2H), 5.36 (s, 2H), 4.93 (t, J = 6.0 Hz, 2H), 4.09 (s, 3H), 3.20 (t, J = 6.0 Hz, 2H). HPLC purity: 95.8%.

Compound 4b. Yellow powder, yield: 62.3%, mp 229-230 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.59 (s, 1H), 8.97 (s, 1H), 8.23 (d, J = 9.2 Hz, 1H), 8.05 (d, J =8.8 Hz, 1H), 7.80 (s, 1H), 7.59 - 7.50 (m, 1H), 7.18 (t, J = 8.0 Hz, 2H), 7.11 (s, 1H), 6.18 (s, 2H), 5.46 (s, 2H), 4.89 (t, J = 5.8 Hz, 2H), 4.02 (s, 3H), 3.20 (t, J = 5.8 Hz, 2H). ESI-MS m/z 448.1 [M - I]+ (Calcd. for C26H20F2NO4+: 448.04). HPLC purity: 93.3%.

Compound 4c. Yellow powder, yield: 67.1%, mp 220- 222 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.70 (s, 1H), 8.92 (s, 1H), 8.22 (d, J = 9.2 Hz, 1H), 8.02 (d, J =9.4 Hz, 1H), 7.78 (s, 1H), 7.52 (d, J =8.1 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.09 (s, 1H), 6.18 (s, 2H), 5.32 (s, 2H), 4.91 (t, J = 6.3 Hz, 2H), 4.09 (s, 3H), 3.20 (t, J =6.3 Hz, 2H), 1.28 (s, 9H). 13C NMR (75 MHz, DMSO) δ 151.44, 151.24, 150.37, 148.22, 145.80, 142.75, 137.91, 134.03, 133.51, 133.42, 131.12, 129.09, 127.21, 125.53, 124.17, 122.34, 120.88, 120.69, 108.91, 105.91, 102.58, 75.76, 57.61, 55.89, 34.82, 31.58, 26.87. ESI - MS m/z 468.1 [M - I]+ (Calcd. for C30H30NO4+: 468.12). HPLC purity: 98.4%.

Compound 4d. Yellow powder, yield: 60.7%, mp 158- 160 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.75 (s, 1H), 8.97 (s, 1H), 8.24 (d, J = 9.2 Hz, 1H), 8.05 (d, J =9.2 Hz, 1H), 7.87 - 7.80 (m, 2H), 7.74 (d, J = 2.0 Hz, 1H), 7.55 (dd, J =8.3, 2.0 Hz, 1H), 7.11 (s, 1H), 6.19 (s, 2H), 5.44 (s, 2H), 4.95 (t, J =5.8 Hz, 2H), 4.06 (s, 3H), 3.20 (t, J = 5.8 Hz, 2H). ESI - MS m/z 480.0 [M - I]+ (Calcd. for C26H20Cl2NO4+: 480.08). HPLC purity: 94.8%.

Compound 4e. Yellow powder, yield: 62.5%, mp 222-224 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.77 (s, 1H), 8.94 (s, 1H), 8.21 (d, J =9.0 Hz, 1H), 8.02 (d, J = 9.3 Hz, 1H), 7.79 (s, 1H), 7.66 (dd, J = 8.3, 5.6 Hz, 2H), 7.23 (t, J =8.7 Hz, 2H), 7.09 (s, 1H), 6.17 (s, 2H), 5.35 (s, 2H), 4.93 (t, J = 5.8 Hz, 2H), 4.09 (s, 3H), 3.20 (t, J = 5.8 Hz, 2H). 13C NMR (75 MHz, DMSO) δ 151.18, 150.36, 148.20, 145.78, 142.42, 137.95, 133.49, 131.63, 131.52, 131.15, 127.10, 124.28, 122.30, 120.89, 120.73, 115.79, 115.51, 108.91, 105.94, 102.58, 75.04, 57.60, 55.87, 26.85. ESI-MS m/z 430.1 [M - I]+ (Calcd. for C26H21FNO4+: 430.05). HPLC purity: 95.7%.

Compound 4f. Yellow powder, yield: 63.3%, mp 158- 160 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.82 (s, 1H), 8.94 (s, 1H), 8.23 (d, J = 8.9 Hz, 1H), 8.04 (d, J =8.9 Hz, 1H), 7.87-7.78 (m, 5H), 7.10 (s, 1H), 6.18 (s, 2H), 5.46 (s, 2H), 4.93 (t, J =6.1 Hz, 2H), 4.07 (s, 3H), 3.21 (t, J = 6.1 Hz, 2H). ESI-MS m/z 480.1 [M - I]+ (Calcd. for C27H21F3NO4+: 480.05). HPLC purity: 96.8%.

Compound 4g. Yellow powder, yield: 60.6%, mp 232- 234 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.86 (s, 1H), 8.96 (s, 1H), 8.30 (d, J = 8.4 Hz, 2H), 8.21 (s, 1H), 8.06 (s, 1H), 7.92 (s, 1H), 7.89 (s, 1H), 7.80 (s, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 5.51 (s, 2H), 4.94 (t, J = 5.8 Hz, 2H), 4.07 (s, 3H), 3.21 (t, J =5.8 Hz, 2H). 13C NMR (75 MHz, DMSO) δ 150.95, 150.39, 148.21, 147.78, 145.69, 144.86, 142.28, 138.09, 133.58, 131.20, 129.79, 129.60, 127.12, 124.53, 123.96, 122.02, 120.89, 120.77, 108.91, 105.96, 102.59, 74.52, 57.64, 55.43, 26.83. ESI-MS m/z 457.1 [M - I]+ (Calcd. for C26H21N2O6+: 457.04). HPLC purity: 91.5%.

Compound 4h. Yellow powder, yield: 63.3%, mp 221- 223 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.94 (s, 1H), 8.23 (d, J = 9.2 Hz, 1H), 8.01 (d, J = 9.1 Hz, 1H), 7.80 (s, 1H), 7.44 (s, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.29 (d, J = 7.5 Hz, 1H), 7.18 (d, J = 7.3 Hz, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 5.31 (s, 2H), 4.93 (t, J = 5.8 Hz, 2H), 4.10 (s, 3H), 3.19 (t, J = 5.8 Hz, 2H), 2.34 (s, 3H). 13C NMR (75 MHz, DMSO) δ 151.26, 150.37, 148.21, 145.78, 142.63, 138.05, 137.92, 136.84, 133.47, 131.14, 129.83, 129.49, 128.72, 127.17, 126.30, 124.22, 122.38, 120.89, 120.71, 108.91, 105.93, 102.58, 75.94, 57.60, 55.87, 26.88, 21.42. ESI-MS m/z 426.2 [M - I]+ (Calcd. for C27H24NO4+: 426.08). HPLC purity: 94.7%.

Compound 4i. Yellow powder, yield: 62.7%, mp 226- 228 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.80 (s, 1H), 8.95 (s, 1H), 8.23 (d, J = 9.2 Hz, 1H), 8.01 (d, J =9.1 Hz, 1H), 7.80 (s, 1H), 7.31 (s, 1H), 7.20 (s, 1H), 7.12 (d, J = 13.0 Hz, 2H), 6.92 (dd, J = 8.2, 1.8 Hz, 1H), 6.18 (s, 2H), 5.34 (s, 2H), 4.93 (t, J = 5.8 Hz, 2H), 4.10 (s, 3H), 3.77 (s, 3H), 3.19 (t, J =5.8 Hz, 2H). 13C NMR (75 MHz, DMSO) δ 159.81, 151.18, 150.37, 148.21, 145.82, 142.53, 138.42, 137.93, 133.47, 131.13, 129.91, 127.16, 124.21, 122.37, 121.34, 120.89, 120.71, 114.61, 114.55, 108.91, 105.93, 102.58, 75.72, 57.61, 55.84, 55.67, 26.87. ESI-MS m/z 442.1 [M - I]+ (Calcd. for C27H24NO5+: 442.07). HPLC purity: 95.5%.

Compound 4j. Yellow powder, yield: 63.1%, mp 194-196 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.71 (s, 1H), 8.92 (s, 1H), 8.22 (d, J =9.3 Hz, 1H), 7.99 (d, J = 9.2 Hz, 1H), 7.79 (s, 1H), 7.51 (d, J =8.5 Hz, 2H), 7.10 (s, 1H), 6.93 (d, J = 8.6 Hz, 2H), 6.18 (s, 2H), 5.30 (s, 2H), 4.92 (t, J = 5.8 Hz, 2H), 4.10 (s, 3H), 3.74 (s, 3H), 3.19 (t, J = 5.8 Hz, 2H). 13C NMR (75 MHz, DMSO) δ 159.94, 151.28, 150.36, 148.21, 145.80, 142.56, 137.86, 133.44, 131.17, 131.11, 128.82, 127.13, 124.09, 122.49, 120.88, 120.69, 114.21, 108.91, 105.91, 102.58, 75.61, 57.58, 55.87, 55.64, 26.87. ESI-MS m/z 442.1 [M - I]+ (Calcd. for C27H24NO5+: 442.07). HPLC purity: 94.4%.

Compound 4k. Yellow powder, yield: 61.8%, mp 226- 227 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.92 (s, 1H), 8.98 (s, 2H), 8.22 (d, J = 9.1 Hz, 2H), 8.02 (d, J =9.2 Hz, 2H), 7.65 (d, J = 8.4 Hz, 1H), 7.47 (s, 1H), 7.10 (s, 2H), 6.18 (s, 2H), 5.35 (s, 2H), 4.95 (t, J = 5.8 Hz, 2H), 4.10 (s, 3H), 3.21 (t, J = 5.8 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 151.09, 150.38, 148.21, 145.76, 142.38, 137.99, 136.02, 133.50, 133.48, 131.16, 131.02, 128.85, 127.12, 124.31, 122.23, 120.89, 120.74, 108.91, 105.94, 102.58, 74.94, 57.60, 55.88, 26.85. ESI-MS m/z 446.1 [M - I]+ (Calcd. for C26H21ClNO4+: 446.02). HPLC purity: 92.7%.

Compound 4l. Yellow powder, yield: 60.1%, mp 229- 231 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.82 (s, 1H), 8.94 (s, 1H), 8.21 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 7.8 Hz, 2H), 7.84 - 7.78 (m, 3H), 7.10 (s, 1H), 6.18 (s, 2H), 5.45 (s, 2H), 4.93 (t, J = 5.8 Hz, 2H), 4.07 (s, 3H), 3.22 (t, J = 5.8 Hz, 2H). 13C NMR (75 MHz, DMSO) δ 150.98, 150.40, 148.22, 145.75, 142.74, 142.28, 138.08, 133.53, 132.82, 131.19, 129.39, 127.13, 124.47, 122.07, 120.88, 120.75, 119.17, 111.41, 108.91, 105.94, 102.59, 74.80, 57.63, 55.88, 26.83. ESI-MS m/z 437.1 [M - I]+ (Calcd. for C27H21N2O4+: 437.05). HPLC purity: 94.0%.

Compound 4m. Yellow powder, yield: 66.2%, mp 158- 160 ℃. 1H NMR (300 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.96 (s, 1H), 8.25 (d, J = 9.2 Hz, 1H), 8.04 (d, J =9.2 Hz, 1H), 7.81 (s, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.31 - 7.22 (m, 3H), 7.10 (s, 1H), 6.19 (s, 2H), 5.35 (s, 2H), 4.90 (t, J = 6.0 Hz, 2H), 4.07 (s, 3H), 3.19 (t, J = 6.0 Hz, 2H), 2.45 (s, 3H). 13C NMR (75 MHz, DMSO) δ 151.39, 150.39, 148.22, 145.63, 142.79, 137.94, 137.22, 135.17, 133.57, 131.13, 130.60, 129.99, 128.97, 127.20, 126.26, 124.34, 122.28, 120.86, 120.78, 108.91, 105.93, 102.58, 74.08, 57.58, 56.42, 26.76, 19.01. ESI-MS m/z 426.1 [M - I]+ (Calc. for C27H24NO4+: 426.08). HPLC purity: 96.6%.

Biological evaluation

BBR and compounds 4a-4m were screened against various bacteria and fungi as per literature protocols [27-28]. All the bacterial strains and fungal strains used in the study were bought from China Center of Industrial Culture Collection. The bacteria and fungi were tested by the broth microdilution method according to guidelines of the Clinical and Laboratory Standards Institute (CLSI) [35-36]. Ciprofloxacin (CIP) was used as a standard drug to compare antibacterial activity whereas Fluconazole (FLU) was used for antifungal evaluation as a reference. Stock solutions of compounds were prepared in DMSO and serially diluted (in geometric progression) in specific medium (final DMSO concentration, 2.5% (V/V). The antimicrobial assay was carried on round-bottomed 96-well plastic microtiter tray and performed in triplicate. The final test concentration of bacteria or fungi was approximately 5 × 105 CFU·mL-1. The final concentrations of test compounds were 1028, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1 µg·L-1, and the final concentrations standards were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 µg·L-1. Incubation was performed at 37 ℃ for 24 h (bacteria) or 25 ℃ for 48 h (fungi). Antimicrobial activity was expressed as MIC (minimal inhibitory concentration, µmol·L-1).

Molecular docking studies

Molecular docking studies were completed by LibDock and CDOCKER module implemented in Discovery Studio (version 3.0, BIOVIA, USA, DS 3.0). The X-ray crystal structure of DNA gyrase-DNA complex with small molecular ligands (PDB ID: 2XCS) [37] was retrieved from the Protein Data Bank. The structure was initially processed by "Prepare Protein" module in DS to give the structures suitable for docking. Missed side chains of the proteins were added and the water molecules were removed, and then the structures were protonated at pH 7.4. "Prepare Ligands" module in DS was applied for the structural preparation of the test compounds, which were then protonated at pH 7.4. The resultant molecules were subsequently minimized by "Minimize Ligands" module. Other parameters were set as default. For molecular docking, the binding site was defined as a site sphere (in 12.7 Å radius) around the original ligands in the co-crystal structures. Other parameters were kept as default. Ten top-ranked conformations for each docked compound were retained.

Conclusions

The present study involved the design, synthesis, and biological evaluation of a series of BBR derivatives with the aim to develop effective and affordable small-molecule antimicrobial drugs and further explain the SAR of the benzyl moiety at C-9. All these newly synthesized products were screened for their in vitro antibacterial and antifungal activities with MIC values in the micromole range. Among them, Compounds 4f, 4g, and 4l showed good anti-microbial activities. It was particularly noteworthy that they showed superior activities against resistant strains (MRSA) than the standard drugs. Furthermore, molecular docking studies showed that our compounds had a strong binding with the protein-DNA complex 2XCS due to their special contact with the target.

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