Synthesis and antibacterial activity of a series novel 5,7-diisoprenyloxyflavone derivatives

*Correspondence: Q.H. Jin. Donghai Science and Technology College, Zhejiang Ocean University, 316000-Zhejiang, Zhoushan, P. R. China. E-mail: qhjin2012@163.com H.Y. Jiang. College of Medicine, Jiaxing University, 314001-Zhejiang, Jiaxing, P. R.China. Tel: +86-580-13957204963. E-mail: jiangyang7689@aliyun.com Synthesis and antibacterial activity of a series novel 5,7-diisoprenyloxyflavone derivatives


INTRODUCTION
According to the World Health Organization (WHO), infectious diseases are responsible for a significant proportion of deaths worldwide, with antimicrobial agents considered "miracle drugs" that are the leading weapons in the treatment of infectious diseases. However, owing to the development of antimicrobial resistance and the appearance of drug-resistant strains among communityacquired infections, there is evidence of the rapid global spread of resistant clinical isolates, with many current clinically efficacious antimicrobial agents becoming less effective. Therefore, the treatment of bacterial infections remains an important and challenging therapeutic problem (Chen et al., 2010). Ongoing drug discovery is necessary for identifying effective, safe, and affordable cures for an expanding spectrum of human ailments. As the treatment of these diseases has serious safety and efficacy issues, the exploration of new antibacterial agents is highly desirable.
Malaria caused by protozoan parasites of the genus plasmodium is a major cause of mortality and morbidity, especially in tropical countries. More than three billion people worldwide have been affected by this deadly disease (Verlinden, Louw, Birkholtz, 2016). The increasing resistance of malarial parasites to available drugs is a major reason for these large statistics, despite many reports on the antimalarial efficacy of flavonoids (Friedman, 2007;Kozyra et al., 2015). The development of novel antimicrobial drugs is still in demand owing to increasing incidence of infection caused by the rapid development of microbial resistance to most known antibiotics.
To obtain new compounds with better antibacterial effects, and as part of our ongoing research on structurebased design using apigenin as the lead compound, the introduction of a prenyloxy group on the A-ring of apigenin was used to prepare a series of 20 novel 5,7-diisoprenyloxyflavone derivatives (4a-4t; Scheme-1). These compounds were synthesized, characterized, and screened for their antibacterial activities. The synthesized of 5,7-diisoprenyloxyflavone derivatives (4a-4t) and antibacterial effects are not reported and is the originality compounds.

Chemistry
Melting points were determined in open capillary tubes and were uncorrected. IR spectra were recorded on a FT-IR1730 (Bruker, Switzerland) using KBr pellets. 1 H-NMR and 13 C-NMR spectra were measured on an AV-300 spectrometer (Bruker, Switzerland), with all chemical shifts given in ppm relative to tetramethylsilane standard. High-resolution mass spectrometry (HRMS) was performed on an MALDI-TOF/TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). Major chemicals were purchased from Aldrich Chemical Corporation (Shanghai, China), and all chemicals were of analytical grade. Compounds 1a-1t, 2a-2t and 3a-3t were synthesized to refer to previously published literature (Guan et al., 2013;Xie et al., 2014.).

General procedure for the preparation of compounds (4a-4t)
To a stirred solution of compounds 3a-3t (0.4 mmol) in DMSO (30 mL) in a 100-mL round-bottomed flask was added I 2 (0.4 mmol). The mixture was then stirred at 100-130 °C for 3-5 h to achieve reaction completion (as monitored by TLC) (Kim et al., 2007). The reaction mixture was then poured into ice-water to produce a yellow precipitate, which was collected and recrystallized from ethanol to afford corresponding products 4a-4t. The yields and IR, 1 H-NMR, 13 C-NMR, and mass spectral data for each compound are provided below.

3-Methoxy
A twofold serial dilution technique (Song et al., 2013) was used to determine the minimum inhibitory concentrations (MICs) of the compounds against susceptible microorganisms in the preliminary test (Grampositive and Gram-negative bacteria) and against strains of clinical isolates of multidrug-resistant Gram-positive bacteria. The compounds dissolved in DMSO and two-fold diluted at concentrations from 200 μg/mL to 0.1 μg/mL, and they were added to culture media (Brain heart infusion for S. mutans and Mueller-Hinton agar for other bacteria) to obtain final concentrations of 0.5-64 μg/mL. The final amount of bacteria applied was 105 CFU/mL. MIC values were determined after incubation at 37 °C for 20 h. The lowest concentration of the test substance that completely inhibited microorganism growth was recorded as the MIC (expressed in μM). Norfloxacin was used as the drug standard. All experiments were conducted in triplicate.

Chemistry
The target compounds were obtained as outlined in Scheme 1. Compounds 1a-1t were synthesized from the Claisen-Schmidt condensation of commercially available 2,4,6-trihydroxyacetophenone (protected as methoxymethyl ethers) with different substituted aromatic aldehydes in aqueous ethanolic sodium hydroxide (Zhen et al., 2016). Intermediates 1a-1t were then treated with 3 M HCl in methanol to yield 2,4,6-trihydroxychalcones derivatives 2a-2t . Subsequent substitution with prenyl bromide in acetone under reflux in the presence of anhydrous K 2 CO 3 afforded compounds 3a-3t (Wang et al., 2015). 5,7-Diisoprenyloxyflavone derivatives 4a-4t were obtained in good yields by treating 3a-3t with I 2 in DMSO. The chemical structures of the target compounds were characterized by IR, 1 H-NMR, 13 C-NMR, and highresolution mass spectroscopy. The IR spectra of the newly synthesized 5,7-diisoprenyloxyflavone derivatives 4a-4t showed absorption stretching bands at 2931-2943 cm -1 and 1672-1678 cm -1 stretching (1220-1222 cm -1 ) corresponding to (-CH 3 ), (C=O) and (C-O-C) group, respectively. IR spectrum of compound 4t showed absorption bands at 3250 cm -1 corresponding to stretching absorption of -OH group. The characteristic feature in the 1 H-NMR spectrum of compound 2t is the appearance of two triplet peaks at 5.41 and 5.44 ppm which represented the prenyl protons in (CH=C-) group. Furthermore, two singlet peaks at 6.73 ppm and 9.12 ppm related to flavone ring 3-C protons in (CH=) group and at phenyl ring 4´-C protons in (-OH) group were observed. 1 H-NMR spectra showed the =CH protons of the group at 6.56-7.02 ppm, The characteristic feature in the 13 C-NMR spectrum of compound 2t displayed four signals peakes in (-CH 3 ) group (19.7 ppm, 19.9 ppm, 25.7 ppm, 25.9 ppm), while showed C=O signals at 182.3 ppm. The structure of 2t was further verified by mass spectroscopy that showed a molecular ion peak [M+H] + at ESI-HRMS 437.1864 (55.4%) in accordance with the molecular formula C 26 H 28 O 6 .

Antibacterial activity
In this study, antibacterial activity was determined from the MIC with different strains in vitro, including multidrug-resistant clinical isolates. Norfloxacin was used as a positive control for bacteria. As shown in Table  I, compounds 4a-4t did not exhibit antibacterial activity against Gram-negative strains at a dose of 24-164 μM in vitro, but some compounds displayed potent antibacterial activity against Gram-positive strains. Nine compounds gave MIC values of 4.4-19 μM. Compounds 4c, 4g, 4i, 4j, 4k, 4l, 4n, 4q and 4t were highly active against S. aureus (S. aureus RN4220, S. aureus KCTC 503,and S. aureus KCTC 209) and Streptococcus mutans KCTC (S. mutans KCTC 3065 and S. mutans KCTC 3289) strains, with MIC values of 4.4-19 μM, but were less active than standard drug norfloxacin. The synthesized derivatives showed significant antibacterial effects against S. aureus, with 2,4-Cl 2 substituted compound 4k giving a MIC value of 4.4 μM, which was similarly active to standard drug norfloxacin.
By analyzing the activities of synthesized compounds 4a-4t, the following structure activity relationships were observed. Eight electron-donor compounds including o-CH 3 , m-CH 3 , p-CH 3 , o-OCH 3 , m-OCH 3 , p-OCH 3 , p-N(CH 3 ) 2 , and 3-OCH 3 -4-OH on the substituent of phenyl ring, were designed and synthesized. Pharmacological test results showed that their activities were lower than those of halogen-substituted derivatives of phenyl ring, with activities in the order 3- For different position methyl and methoxy groups on the substituent of phenyl ring influenced the antibacterial effects with activities in the order m-CH 3 > o-CH 3 , p-CH 3 , and m-OCH 3 > o-OCH 3 , p-OCH 3 . Furthermore, the position of electron-withdrawing (F, Cl, and Br) groups on the B ring (of phenyl ring) significantly influenced the antibacterial activity, with activities in the order m-F > p-F > o-F for fluoro-substituted compounds of phenyl ring, and p-Br > m-Br > o-Br for bromo-substituted compounds of phenyl ring. In comparison, the chloro-substituted derivatives of phenyl ring showed activities in the order 2,4-dichloro > p-Cl > m-Cl > o-Cl. Therefore, compounds 4c, 4g, and 4j bearing m-F, p-Cl, and p-Br substituents of phenyl ring, respectively, showed better activities, while those bearing o-F, o-Cl, and o-Br substituents of phenyl ring (4b, 4e, and 4f, respectively) were inactive for all microorganisms, even at doses of 24-151 μM. Compound 4k (MIC = 4.4 μM) was 30-fold more potent than apigenin (MIC = 118.5 μM). It has been proposed that the prenyl moiety on A ring makes compounds more lipophilic, which leads to a higher affinity with cell membranes, with prenylation shown to afford flavonoids with enhanced antibacterial activities (Ei-Bassuony, Abouzid, 2010;Yu et al., 2015).

Activity against clinical isolates of multidrugresistant Gram-positive bacteria
The most active compounds, 4c, 4g, 4i, 4j, 4k, 4l, 4n, 4q and 4t, were also evaluated for antibacterial effects against clinical isolates of multidrug-resistant Grampositive bacteria (Table II). These derivatives were found to be highly active against these clinical isolates, giving MIC values of 4.0-20 μM. Compound 4k was more potent than norfloxacin against most microorganisms tested, giving an MIC value of 4.0 μM. This suggested that the introduction of two halogen atoms of phenyl ring and a prenyl moiety on A ring into the hybrid compound played an important role in improving the antibacterial properties (Chen et al., 2014;Marín, Máñez, 2013). Therefore, compound 4k should be used as the lead compound for further design and investigations.

CONCLUSION
We synthesized a series of novel 5,7-diisoprenyloxyflavone derivatives and evaluated their antibacterial effects against Gram-positive and Gram-negative bacteria.
Compounds 4c, 4g, 4i, 4j, 4k, 4l, 4n, 4q and 4t were highly active against S. aureus and S. mutans KCTC, giving MIC values of 4.0-20 μM, and also showed high activities against clinical isolates of multidrug-resistant Gram-positive bacteria, with MIC values of 4.0-20 μM. In particular, compound 4k was more potent than norfloxacin against most microorganisms tested, giving a better MIC value of 4.0 μM. This indicated that hybrid compounds possessing flavone and prenyl moieties might possess improved antibacterial properties. These results indicate that the further design and development of such compounds will be of interest in future research.