Preparation and antibacterial activity of coronarin E derivatives

Abstract Coronarin E is a main diterpene ever isolated from Hedychium yunnanense. With the aim to enlarge its potential application, four butenolide derivatives (compounds 4a, 4b, 5a and 5b) were obtained from coronarin E via synthetic method, and their antibacterial effects were also evaluated. It is noteworthy that compounds 5a and 5b exhibited stronger antibacterial activities against most of the tested bacterial strains than ampicillin and kanamycin, two first- and second-line antimicrobials in clinical. For example, minimum inhibitory concentration (MIC) of 5a, 5b, ampicillin and kanamycin against Acinetobacter baumanii were 2, 1, 8 and 4 μg/mL, respectively, and MIC of the four compounds mentioned above against Klebsiella pneumonia were 1, 0.5, 16 and 4 μg/mL, respectively. The current studies not only enrich the structural diversity of diterpenes derived from Hedychium genus, but also provide potent candidates for the development of antibacterial medicines. GRAPHICAL ABSTRACT


Introduction
Abuse of antibiotics has led to more and more serious antibiotic resistance of pathogens in the past decades.Drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) strains and Acinetobacter baumanii, pose great menace to human health worldwide (Liu et al. 2016;Zuo et al. 2018).Therefore, it is urgent to search for new antimicrobial agents from natural products or their synthetic derivatives.
Recently, a number of diterpenoids isolated from Zingiberaceae family in recent years revealed significant antimicrobial activities (Morita and Itokawa 1988;Chen et al. 2010;Ghosh et al. 2013;Sivasothy et al. 2013;Yu et al. 2015;Zhao et al. 2018).For example, coronarin D, a diterpenoid isolated from Hedychium coronarin displayed promising antimicrobial property against Candida albicans.(Kaomongkolgit et al. 2012).Based on the observed antimicrobial activities for 15 terpenoids, some Chilean scholars put forward a hypothesis that antimicrobial activities of the terpenoids were composed of two structural requirements: a hydrophobic moiety, together with a hydrophilic region.Such amphiphilic molecular could readily insert into phospholipid bilayer of microbial cell-membranes (urzúa et al. 2008).Coronarin E (Zhao et al. 1995) was an abundant diterpene found in some species of Zingiberaceae family, such as Hedychium yunnanense.Inspired by aforementioned information, we planned to introduce an aromatic acetoxy group into B-ring of coronarin E firstly, and then oxidize the furan ring to construct butenolide ring.The final products composed a hydrophobic moiety (A, B ring of the diterpene and the aromatic acetoxy group), and a hydrophilic region (butenolide ring).Such amphiphilic products may be effectively insert into phospholipid bilayer of bacterial membranes and finally damaged the membranes.Just as expected, four final products, 4a, 5a, 4b, and 5b showed strong antibacterial activities against several bacterial cells.herein, the structural transformation of coronarin E and the antibacterial effects of obtained derivatives are reported.

Results and discussion
Coronarin E was oxidized with Seo 2 and t butyl hydroperoxide to obtain compound 1 (Scheme 1), which underwent acylation and subsequent photosensitized oxidation to obtain compounds 4a/5a and 4b/5b (Scheme 2).Some 13 C or 1 h signals of 4a and 4b were shown to split, indicating that both 4a and 4b might be mixtures of two epimers.Two epimers can transform from one to another rapidly in solution (Scheme 3).The transformations of these two epimers were so rapid that they could not be effectively separated.As a result, 4a and 4b behaved as a single compound chromatographically.Theoretically, although both 5a and 5b should be mixtures, all 13 C signals of 5a and 5b did not split in solution, which could be due to the long distance between the C-15 hydroxyl group of 5a or 5b at and A/B ring moiety of diterpene skeleton.Compounds 4a, 5a, 4b, and 5b were tested for their antibacterial activities by a conventional agar-dilution method.The minimum inhibitory Scheme 1. Preparation of compound 1. concentration values (MICs) of the four compounds were summarized as shown in Table 1.Pharmacologically, the four compounds showed promising antibacterial activities.Interestingly, both 5a and 5b displayed broader spectrum antibacterial properties than 4a and 4b.Furthermore, 5a and 5b were discovered to have potent antimicrobial activity against the majority of tested gram-positive and gram-negative bacteria, with particularly potent inhibition activity against Staphylococcus aureus, MRSA1450, MRSA 1505, MRSA 1591, MRSA2024, MRSA 1957, MRSA I-20, MRSA I-67, Streptococcus pneumonia, Enterococcus faecalis, Escherichia coli, Acinetobacter baumanii, Klebsiella pneumoniae, and Haemophilus influenzae, and their activities against most of the bacteria mentioned above were higher than those of ampicillin, and kanamycin, two first-and second-line antimicrobials in clinical.Furthermore, 4a and 4b inhibited Escherichia coli, MRSA1450, MRSA 1505, MRSA 1591, and Haemophilus influenza effectively.
Based on the data shown in Table 1, compounds 5a and 5b were subjected to drug combination test with three antibiotics, vancomycin, ampicillin and kanamycin, respectively.Checkerboard methods were utilized in the combination test, and the results were shown in Tables 2 and 3. Compound 5a showed synergy with vancomycin against H. influenza, with FICI of 0.5.Compound 5b showed synergy with kanamycin against K. pneumonia, with FICI of 0.5.Furthermore, 5a and 5b had additive effect against most of the bacterial strains when combined with vancomycin, ampicillin or kanamycin.
Coronarin E, a labdane-type diterpene from Zingiberaceae plant, was converted to four butenolide derivatives in this study.Products 5a and 5b showed good antibacterial activities against various gram-positive bacteria and gram-negative bacteria.It is noteworthy that 5a and 5b revealed very good antibacterial activity against A. baumanii and K. pneumonia, which were very troublesome gram-negative bacteria in clinic (Especially A. baumanii ranked among the Who priority list of antibiotic-resistant pathogens in 2018 (Tacconelli et al. 2018)), and their inhibitory activities were higher than those of ampicillin and kanamycin.Besides, 5a had a synergistic action with vancomycin against H. influenza, while 5b had a synergistic action with kanamycin against K. pneumonia.This study revealed a new way for the development of antimicrobial medicines.Furan-type diterpenoids were abundant in Zingiberaceae family, which may be pretty good starting materials of synthetizing potent antibacterial amphiphilic molecules as follows: First, aromatic acetoxy group was introduced into B-ring of diterpenoids to construct hydrophobic moiety; Second, furan ring was oxidized to construct butenolide ring, a hydrophilic group.

General experimental procedures
NMR spectra were recorded using a Bruker Avance III spectrometer (500 Mhz for 1 h NMR and 125 Mhz for 13 C NMR) with TMS as an internal standard.TLC was performed on silica gel 60 F 254 or silica gel 60 RP-18 F 254 (Merck, Germany), and the spots were visualized by spraying with a 10% h 2 So 4 solution and then heating.Silica gel was used for column chromatography (300-400 mesh; Qingdao Marine Chemical Co., Qingdao, China).

Preparation of coronarin E
The  The air-dried and powdered rhizomes of H. yunnanense (2.0 kg) were extracted with 95% ethanol three times to obtain a crude extract.Then the ethanol extract was suspended in h 2 o, and extracted with chloroform three times.After removal of the solvent, the extract (64 g) was subjected to silica gel column with a gradient of petroleum ether-EtoAc (1:0→0:1, v/v) to afford 8 fractions (Fr.1-Fr.8).Fr.1 was separated using silica gel column eluting with petroleum to afford 3 fractions (Fr.1-1∼Fr.1-3).Fr.1-3 was crystalized with petroleum at 0 °C to obtain 2.1 g of coronarin E as needles.

General method for synthesis
Preparation of compound 1 (Miyake et al. 2008): 168 mg of selenium dioxide, 368 mg of 60% tert-butyl hydroperoxide, and 5 mL of dried Ch 2 Cl 2 were introduced into a round-bottomed flask, followed by 400 mg of coronarin E. The resulting mixture was stirred at room temperature for 2 h.The reaction mixture was subjected to silica gel column chromatography eluting with petroleum ether/ethyl acetate gradient elution (80:1→40:1) to obtain compound 1, yielding 300 mg (71%).
Preparation of 3a: 208 mg of p-isopropyl benzoic acid (2a), 11 mg of 4-dimethylaminopyridine (DMAP), and 298 mg of N, N-diisopropylcarbodiimide (DIC), and 6 mL of dried Ch 2 Cl 2 were introduced into a round-bottomed flask and stirred for 30 min at room temperature.Subsequently, 150 mg of compound 1 was added and the solution was stirred for 5 h.The reaction mixture was quenched with 1.0 mL of water.10 mL of 70% methanol was added and the solution was extracted with petroleum ether (15 mL × 2).The combined petroleum ether extract was condensed, and the residue was subjected to silica gel column chromatography eluting with petroleum ether/chloroform gradient elution (8:1→1:1) to give 3a (145 mg, yield 65%). Preparation of 3b: 210 mg of p-butyl benzoic acid (2b), 11 mg of DMAP, 300 mg of DIC, and 6 mL of dried Ch 2 Cl 2 were introduced into a round-bottomed flask and stirred for 30 min at room temperature.Subsequently, 150 mg of compound 1 was added and the solution was stirred for 5 h.The reaction mixture was quenched with 1.0 mL of water.10 mL of 70% methanol was added and the solution was extracted with petroleum ether (15 mL × 2).The combined petroleum ether extract was condensed, and the residue was subjected to silica gel column chromatography eluting with petroleum ether/chloroform gradient elution (8:1→1:1), compound 3b (146 mg) was obtained, yielding 64%.
Preparation of compounds 4a and 5a: 127 mg of compound 3a, 2.0 mg of tetraphenylporphyrin (TPP), and 5.0 mL of dried pyridine were introduced into a round-bottomed flask.The solution was stirred and illuminated with an LED lamp for 8 h at room temperature.The solution was concentrated and the residue was purified using silica gel column chromatography eluting with petroleum ether and ethyl acetate (3:1→1:1).Compounds 4a (36 mg, yield 26%) and 5a (24 mg, yield 18%) were obtained consequently.
Preparation of compounds 4b and 5b: 120 mg of compound 3b, 2.0 mg of TPP, and 5 mL of dried pyridine were introduced into a round-bottomed flask.The solution was stirred and illuminated with an LED lamp for 8 h at room temperature.The solution was concentrated and the residue was purified using silica gel column chromatography eluting with petroleum ether and ethyl acetate (3:1→1:1).Compounds 4b (29 mg, yield 22%) and 5b (21 mg, yield 16%) were obtained consequently.

Conclusion
In summary, four butenolide derivatives (compounds 4a, 4b, 5a and 5b) were obtained from coronarin E, a main diterpene ever isolated from Hedychium yunnanense, via synthetic method.Compounds 5a and 5b exhibited stronger antibacterial activities against most of the tested bacterial strains than two first-and second-line antimicrobials in clinical, ampicillin and kanamycin.
'-': means that the data was not measured; vanc, ampi, and kana in the table mean vancomycin, ampicillin, and kanamycin, respectively.

Table 2 .
combination test of 5a with three antibiotics.

Table 3 .
combination test of 5b with three antibiotics.