Design, synthesis, and antimicrobial activities of new tanshinone IIA esters

Abstract Sixteen new ester derivatives with various partition coefficient (ClogP) values of tanshinone IIA (TSIIA), a major lipophilic component of Salvia miltiorrhiza, were designed and synthesised, including six aliphatic esters (3a–e, 5a), one phosphate ester (4c) and nine aromatic esters (5b–j). Their antimicrobial activities against three Gram-positive bacteria strains, Staphylococcus aureus, Bacillus subtilis, and Bacillus amyloliquefaciens, and two Gram-negative bacteria strains, Pseudomonas aeruginosa and Escherichia coli, as well as two fungi species, Candida albicans and Saccharomyces cerevisiae, were evaluated in vitro by broth microdilution susceptibility tests. The results showed that keeping ClogP values in a certain range is necessary for their antimicrobial activities. For those compounds with ClogP values between 5 and 10, their MIC values showed positive correlations with ClogP values. In particular, compound 3e exhibited fourfold and twofold higher potency than the standard drug amphotericin B against fungi C. albicans and S. cerevisiae with MIC values of 1.95 and 7.81 μg/mL, respectively. Graphical abstract


Introduction
Infections have been a serious and growing threat due to increasing multidrug resistance (MDr) of microbial pathogens (Bancroft 2007), especially when very few new drugs have been brought to market after older classes lose their efficacy (Spellberg et al. 2008;Hogberg et al. 2010). Natural products or compounds derived from natural structures present advantages to overcome MDr and may hopefully be candidates for new antimicrobials (King et al. 2014;Sheng et al. 2014). Tanshinone IIA (TSIIA), a major lipophilic component of the well-known traditional Chinese medicine Salvia miltiorrhiza, has showed antimicrobial, antioxidant, antitumor, cardiovascular protective and hepatoprotective activities (Li et al. 2013;Huang et al. 2015;Maione et al. 2015). A hydrophilic derivative of TSIIA, sodium tanshinone IIA sulfonate (STS), has been approved for clinical usage to cure cardiovascular disease in China in the 1980s (Tian & Wu 2013). STS significantly improved the poor water solubility of TSIIA with the strategy to introduce an ionic polar group at α-position of the furan D ring and showed similar cardiovascular activity as that of TSIIA. However, the antibacterial effect of STS is much weaker than that of TSIIA in our preliminary screening, probably due to the hydrophobic barrier of cellular membrane. The results also indicate that structure modification to maintain the lipophilic character of TSIIA in a certain range is crucial for its antibacterial effect. Following this concept and using the calculated lipid water partition coefficient (ClogP) as an index, 16 new ester derivatives of TSIIA with enhanced or decreased lipophilicities were designed and synthesised, including six aliphatic esters (3a-e, 5a), one phosphate ester (4c), and nine aromatic esters (5b-j). Their antimicrobial activities against three Gram-positive bacteria strains, Staphylococcus aureus, Bacillus subtilis, and Bacillus amyloliquefaciens and two Gramnegative bacteria strains, Pseudomonas aeruginosa and Escherichia coli, as well as two fungi species, Candida albicans and Saccharomyces cerevisiae, were evaluated in vitro by broth microdilution susceptibility tests.

Results and discussion
It is known that lipophilicity plays an important role in producing antibacterial effect as related to membrane permeation in biological system (Ansari & Lal 2009). In order to investigate possible relationship between lipophilicities and antimicrobial activities of TSIIA esters, 16 derivatives were designed by introducing different polar ester groups, including saturated and unsaturated aliphatic acids, phosphate, and aromatic acids, into the quinone moiety of TSIIA. General approaches to synthesise a variety of ester derivatives of TSIIA were outlined in the schemes as illustrated.
Starting from TSIIA (1), the intermediate hydroxytanshinone IIA (2) was prepared through reduction of 1 under hydrogen/palladium carbon. As the resulting compound 2 is unstable in air due to rapidly auto-oxidation, anhydride or acyl chloride were subsequently added to the solution of 2 under inert gas to achieve the desired compounds 3a-3e, 4a-4c, and 5a-5j, respectively. Compounds 3a-3e were synthesised with anhydride and 4-dimethylaminopyridine (DMAP) in anhydrous tetrahydrofuran (Scheme 1). Compound 4c was prepared in two steps by two methods. Firstly, diethyl chlorophosphate or dibenzylphosphoryl chloride was used to obtain the intermediates 4a and 4b, respectively. In the second step, 4c was acquired through deprotection of 4a with bromotrimethylsilane or by hydrogenolysis of the benzyl protecting groups of 4b, followed by neutralisation with sodium methoxide (Scheme 2). Treatment of 2 with different aromatic acids or the aliphatic methacrylic acid in the presence of DMAP, N-methylmorpholine (NMM) and pivaloyl chloride gave compounds 5a-5j (Scheme 3).
The microdilution susceptibility test in Luria-Bertani (LB) broth and yeast extract peptone dextrose (YePD) medium were carried out for the determination of antibacterial and antifungal activities, respectively. Antimicrobial activities of 17 compounds (1, 3a-3e, 4c and 5a-5j) were evaluated in vitro against three Gram-positive bacteria strains, S. aureus, B. subtilis and B. amyloliquefaciens, and two gram-negative bacteria strains, P. aeruginosa and E. coli, as well as two fungal strains, C. albicans and S. cerevisiae. Minimum inhibitory concentration (MIC) for the tested compounds was determined by using the micro-well serial dilution method according to the literature (Challa et al. 2014 The MIC values of these compounds against the tested bacteria and fungi were summarised in Table 1. Compounds were listed in the order of their ClogP values predicted by ChemBioDraw ultra 13.0 software package. TSIIA (1, ClogP = 5.74) exhibited broad-spectrum and moderate antimicrobial activities against the test microorganisms with MIC values of 31.25-125 μg/mL. Both the most hydrophilic molecule 4c (ClogP = 2.98) and the most lipophilic esters (5e-i, ClogP > 10) showed no activity, which indicate that a certain range of hydrophilic/lipophilic property is necessary for the antimicrobial activity. For those compounds with ClogP values between 5 and 10, their MICs showed positive correlation with their predicted ClogP values. Compounds 3a and 3e with slightly lower ClogP values exhibited improved antibacterial activities in almost all microorganism species. In particular, compound 3e has showed fourfold more active and twofold higher potency than the standard antifungal drug amphotericin B (MIC = 7.81 and 15.63 μg/mL, respectively) against C. albicans and S. cerevisiae with MIC values of 1.95 and 7.81 μg/mL, respectively. our results Scheme 3. synthesis of compounds 5a-j also brought new hope to treat C. albicans infections that contribute over 60% invasive candidiasis clinical infections and mortality of 15-35% for adults and 10-15% for neonates (Guinea 2014).

General
TSIIA was purchased from Xi'an Guanyu Bio-technique Co. Ltd with purity ≥98%. reagent grade chemicals were purchased from commercial supplier and used without purification. All the solvents were dried by standard methods in advance and distilled before use. reactions were performed under an inert atmosphere using nitrogen gas, unless specified. Thin-layer chromatography (TLC) plates (GF254, Merck) were used to monitor reactions. Purification of intermediates and products were performed by silica gel (200−400 mesh, 60 Å) column chromatography. Melting points were measured on an X-4 electro thermal melting point apparatus. NMr spectra were recorded on Varian Inova-400 and Bruker Advance spectrometers (500 MHz, 600 MHz) with TMS as internal standard. Chemical shifts were expressed in ppm, and coupling constants (J) were given in Hz. Mass spectra were recorded on an esquire-LC 00075 spectrometer and the signals were given in m/z.

Synthesis of TSIIA derivatives
A mixture of TSIIA (1 mmol) and 10% Pd/C (30 mg) in anhydrous tetrahydrofuran (THF, 10 mL) was stirred under hydrogen gas at room temperature until red color of the solution faded, indicating that the ketone groups of 1 were reduced thoroughly to hydroxyls of compound 2. A solution of DMAP (60 mg, 20%) and anhydride (2.5 mmol) in 5 mL of THF were then added dropwisely into the reaction mixture under N 2 in an ice bath. The reaction mixture was stirred at room temperature for 12 h, followed by filtration of Pd/C and evaporation of the solvent under reduced pressure. The residue was further purified by column chromatography eluted with petroleum ether/ethyl acetate (φ r = 9:1) followed by recrystallisation to get the products 3a-3e in good yields (≥80%) (Scheme 1).
To a stirred solution of compound 2 (1 mmol) in anhydrous THF (10 mL) in an ice bath, NaH and diethyl chlorophosphate or dibenzylphosphoryl chloride (2.2 mmol) were added orderly under argon gas. The reaction mixture was then slowly warmed up to room temperature and stirred overnight. Afterwards, the mixture solution was filtered and diluted with 20 mL of water, followed by over three times of extraction with ethyl acetate. The combined organic layers were dried by adding anhydrous Na 2 So 4 and evaporated under reduced pressure. The crude products were purified by column chromatography eluted with hexane/acetone (φ r = 9:1) to give the desired intermediates 4a and 4b, respectively. The terminal salt 4c was obtained by deprotection of 4a with bromotrimethylsilane or by hydrogenation of 4b with palladium carbon and hydrogen gas, followed by neutralisation with sodium methoxide in almost quantitative yields (Scheme 2).
Commercially available carboxylic acids (3 mmol) were dissolved in anhydrous THF (10 mL), N-methyl morpholine and pivaloyl chloride (each 3 mmol) were added orderly under an inert atmosphere using nitrogen gas. The stock solution was then allowed to stir at −10 °C for 1 h and was added directly, along with DMAP (60 mg, dissolved in 5 mL of THF), to a stirred solution of compound 2 (1 mmol) in anhydrous THF (10 mL). After 30 min, the reaction mixture was slowly heated to reflux temperature and maintained for 6 h (Scheme 3). The solution was filtrated and evaporated under reduced pressure to give a residue that was subsequently purified by silica gel column chromatography eluted with petroleum ether/ethyl acetate (φ r = 9:1) followed by recrystallisation to afford the desired products 5a-5j (Scheme 3). For spectroscopic data of compounds 3a-e, 4a-c and 5a-j, see supplementary material.

Antimicrobial activity assay
The microdilution susceptibility test in LB broth and YePD medium were carried out for the determination of antibacterial and antifungal activities, respectively. Antimicrobial activities of 17 compounds (1, 3a-3e, 4c and 5a-5j) were evaluated in vitro against three Gram-positive bacteria strains, S. aureus (ATCC-25923), B. subtilis (ATCC 530) and B. amyloliquefaciens (CC09), and two Gram-negative bacteria strains, P. aeruginosa (ATCC-27853) and E. coli (ATCC-25922), as well as two fungal strains, C. albicans (ATCC-10231) and S. cerevisiae (ATCC-204508). MIC for the tested compounds was determined by using the microwell serial dilution method according to the literature (Challa et al. 2014).
Briefly, the tested compounds were dissolved in DMSo (500 μg/mL) and 100 μL of the solution were transferred to a 9-mm-diameter hole punched in a LB or YePD agar plate seeded with the appropriate test organisms. Ciprofloxacin and amphotericin B were used as positive controls for antibacterial and antifungal experiments, respectively, while DMSo was used as a blank control. The plates were incubated at 37 °C for 24 h and 30 °C for 48 h to culture the bacteria and fungi, respectively. The zone of inhibition (expressed in mm) results represented the antimicrobial potency. All the assays were carried out in triplicates. Compounds that showed significant growth inhibition zones (≥14 mm) were further evaluated for their minimal inhibitory concentrations (MICs) using the twofold serial dilution technique in 96-well microtiter plate. Specifically, inoculum size for test strains were adjusted to 1 × 10 6 CFu/mL, and 100 μL microorganism suspensions were transferred to each well. Stock solutions of the tested compounds were prepared in DMSo at 1000 μg/mL followed by twofold dilution at concentrations of 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 μg/mL. The prepared solutions (100 μL) ranging from 250 to 0.49 μg/mL were thereafter transferred to the wells. As an indicator, 10 μL of 5 mg/mL 2,3,5-triphenyltetrazolium chloride (TTC) was added at 20 h for the bacteria and at 44 h for the fungi, respectively.

Conclusion
In conclusion, 16 ester derivatives of TSIIA with a broad spectrum of ClogPs were designed and synthesised, among which a new compound 3e exhibited higher level of antifungal activity than the standard drug amphotericin B. The results also indicated that a certain range of hydrophilic/lipophilic property (ClogP) is necessary for the antimicrobial activity. In that range, MIC values of those compounds showed positive correlations with their ClogP values and TSIIA ester derivatives with improved hydrophility may have higher level of antimicrobial activity.