Design, Synthesis and Evaluation of the Antibacterial Enhancement Activities of Amino Dihydroartemisinin Derivatives

Artemisinin (ART) and its derivatives artesunate (AS), dihydroartemisinin (DHA) are a group of drugs containing a sesquiterpene lactone used to treat malaria. Previously, AS was shown to not have antibacterial activity but to significantly increase the antibacterial activities of β-lactam antibiotics against E. coli. Herein, molecular docking experiments showed that ART, AS and DHA could dock into AcrB very well, especially DHA and AS; both DHA and AS had the same docking pose. The affinity between AS and AcrB seemed weaker than that of DHA, while the succinate tail of AS, which was like a “bug”, could extend in the binding pocket very well. Imitating the parent nucleus of DHA and the succinate tail of AS, twenty-one DHA derivatives 4a–u were designed and synthesized. Among them, seventeen were new compounds. The synergistic effects against E. coli AG100A/pET28a-AcrB showed among the new structures 4k, 4l, 4m, 4n, and 4r exhibited significant synergism with β-lactam antibiotics although they had no direct antibacterial activities themelves. The bacterial growth assay showed that only 4k in combination with ampicillin or cefuroxime could totally inhibit bacterial growth from 0 to 12 h, demonstrating that 4k had the best antibacterial enhancement effect. In conclusion, our results provided a new idea and several candidate compounds for antibacterial activity enhancers against multidrug resistant E. coli.


Computer-Predicted Ligand Binding to AcrB
To investigate whether and how ART, DHA and AS bound AcrB, molecular docking experiments were carried out based on a previous report [13,14]. Herein, the reported AcrB crystal structure was used as the rigid "receptor", and the binding to the receptor of three candidate ligands which were set as flexible conformations was examined by the MOE program. Because the three candidate ligands had same parent sesquiterpene lactone structure, the three were put into the same domain of AcrB to trigger the docking processes. The London dG scores of ART, DHA and AS were −13.28, −14.76 and −15.02, respectively. The above docking results suggested that the three candidate ligands could dock into AcrB very well, especially DHA and AS. In the most favored docking mode, the three ligands were revealed to be all bound to the upper portion (farther away from the membrane surface) of the drug-transport pocket ( Figure 1). The shape of upper portion was like a diminishing "cave", forming a small drug-transport tunnel. Therefore, ART, DHA and AS could be defined as cave-binders and supposed to block the drug efflux. Although the three ligands had the same parent sesquiterpene lactone structure, only DHA and AS had same docking poses, while ART was different from its two derivatives ( Figure 1). The parent sesquiterpene lactone structure of DHA could tightly bind to Ser46, Val88, Gln89, Gln176 and Arg620, which were around the entrance surface to the "cave". DHA formed seven hydrogen bonds via the four heterocyclic oxygen atoms of the parent structure with the residues mentioned above, so the ligands seemed to cap the "cave" entrance. The parent structure of AS similarly bound to the entrance surface of the "cave", forming five hydrogen bonds with Ser46, Gln89 and Gln176.
AcrB contains three subdomains, including a TolC-docking subdomain (located in the N-terminal end) and a porter (middle) of the periplasmic domain, as well as a transmembrane domain (located in the C-terminal end) which were also identified before [14]. A drug-transport pocket involved in the porter had been defined as the main substrate efflux tunnel [9,15]. The binding between AS and AcrB seemed weaker than that of DHA, while the succinate tail of AS, like a "bug", could extend in the binding pocket very well. This might help AS block the drug efflux and confer antibacterial enhancement effects. The parent structure of ART could only form three hydrogen bonds with Ser46 and Arg620, and ART had no "bug" like AS, so the binding between AS and AcrB was weaker than between DHA and AS. The above docking results suggested the structures of DHA and AS were good references to obtain more effective EPIs.

Synthesis of DHA Derivatives
Based on above results and suggestions, twenty-one DHA derivatives 4a-u were designed in order to imitate the parent nucleus of DHA and the succinate tail of AS, and then the derivatives were synthesized. The synthetic route to the DHA derivatives is illustrated in Scheme 1. Among these derivatives, seventeen were new ( Figure 2 Figure 2. The structures of 17 new compounds.

DHA Derivatives Have no Directly Antibacterial Activity
The results showed E. coli AG100A/pET28a-AcrB was resistant to ampicillin and cefuroxime, with MICs of 2 and 512 μg/mL, respectively. The MICs of most of the DHA derivatives were more than 1,024 μg/mL, and some were more than 2,048 μg/mL (Table 1), which was thought to be no clinically significance since the MIC was so high, and suggesting these DHA derivatives had no direct antibacterial activity. The results from the drug susceptibility assay showed that the fractional inhibitory concentration index (FICI) values produced by five DHA derivatives (compounds 4k, 4l, 4m, 4n, and 4r) in combination with ampicillin and cefuroxime were less than or equivalent to 0.5 ( Table 2), demonstrating that these five DHA derivatives could increase the antibacterial activities of ampicillin and cefuroxime. Importantly, these five DHA derivatives in combination with ampicillin and cefuroxim produced a lower FICI, suggesting these derivatives possibly had stronger antibacterial enhancement activities. Among these five DHA derivatives, 4k, 4l, and 4m possessed more significant enhancement for both ampicillin and cefuroxime; FICIs were lower than 0.2 (Table 2). Therefore, these three derivatives were further investigated in subsequent experiments to observe their influence on bacterial dynamic growth. Table 2. FICI values for DHA derivatives in combinations with ampicillin and cefuroxime against E. coli AG100A/pET28a-AcrB. Synergistic effects of different concentrations of DHA derivatives in combination with ampicillin and cefuroxime were evaluated using chequerboard method. FICI values were interpreted as follows: <0.5 = synergy; 0.5-4.0 = no interaction; and >4.0 = antagonism. AMP, ampicillin; CFX, cefuroxime. The results from dynamic bacterial growth assay showed E. coli AG100A/pET28a-AcrB grew very well even though antibiotics were added. However, bacterial growth was inhibited by 4k, 4l, and 4m in combinations with antibiotics during the exponential phase of growth (from 3 to 9 h) although these three derivatives had no such an effect (Figure 3) alone. Interestingly, the bacterial growth was totally inhibited only by 4k in combination with ampicillin or cefuroxime from 0 to 12 h; the OD 600 value was obviously lower than that of antibiotics alone, demonstrating 4k had the best antibacterial enhancement effect and could be considered as a good EPI candidate.
As well-known, decreased accumulation of antibiotic is the main multidrug resistance mechanism for Gram-negative bacteria. Previously, AS was first found to increase drug accumulation in a dosedependent manner, suggesting the antibacterial enhancement of AS was tightly associated with increased antibiotic accumulation via targeting of AcrB because AS lost its enhancement activities against AcrB-knocked out E. coli. Therefore, AS was thought to be an EPI [12]. Herein, based on the results of molecular docking experiments, twenty-one DHA derivatives were designed and synthesized to imitate with parent nucleus of DHA and succinate tail of AS. Among them, 4k, 4l, 4m, 4n, and 4r with new structures exhibited significant synergism with β-lactam antibiotics although they had no directly antibacterial activities themselves. The above results demonstrated these DHA derivatives possessed antibacterial enhancement activities just like AS, suggesting these DHA derivatives were worth further investigation as EPIs.
Considering these DHA derivatives were designed to imitate the parent nucleus of DHA and the succinate tail of AS and they had similar activities to AS, the possible molecular mechanism of these DHA derivatives was thought to also target AcrB to block the AcrAB-TolC efflux pump, leading to increased accumulation of antibiotic. Of course, a series of experiments should be done in the subsequent investigation to prove this. Influence of DHA derivatives on dynamic growth curves of E. coli AG100A/pET28a-AcrB. The bacteria from the exponential phase of growth were diluted with LB broth to 1.0 × 10 6 cfu/mL. Indicated concentrations of DHA derivatives and antibiotics (1/2 MIC) were added into bacterial suspension. Bacterial growth was determined by measuring OD 600 at regular intervals. AMP, ampicillin; CFX, cefuroxime; 4k, 4l, and 4m, DHA derivatives (256 μg/mL, <1/2 MIC) in combinations with AMP (8 μg/mL, 1/2 MIC) or CFX (128 μg/mL, 1/2 MIC). Representative data from one of three independent experiments are shown; the standard deviation bars are not shown. Wuling Wountain Pharmaceutical Corporation, Ltd, Chongqing, China). Other reagents were all analytically or chemically pure compounds bought from the market and not further processed.

Bacterial Strains
E. coli AG100A lacking the gene encoding AcrAB was donated by Professor Hiroshi Nikaido of the University of California (Berkeley, CA, USA). E. coli AG100A/pET28a-AcrB re-expressing AcrB, the recombinant AG100A harboring pET28a-AcrB, was constructed in our laboratory.

Molecular docking
The crystal structure of AcrB [7] (PDB ID, 1IWG) was obtained from the Protein Data Bank, and the three-dimensional structural models of ART, DHA, and AS were drawn in Chem3D Ultra. The molecular docking was performed in MOE 2008 using the Triangle Matcher approach. In the docking calculations of the MOE program, AcrB was defined as the receptor, ART, DHA, and AS were respectively defined as the ligands, other parameters during the docking process were set at default values. Quality assessment of the models was performed using London dG. (3) 2-Bromoethyl alcohol (23.103 g, 24 mmoL) and Et 2 O (100 mL) were placed into a 250 mL round bottomed flask, and then BF 3 .Et 2 O (4 mL) was added under ice bath cooling. Dihydroartemisinin (15.690 g, 20 mmol) was finally added with stirring. The mixture was stirred for 1.5 h and continuously ice bath cooled. The reaction progress was monitored with TLC. Saturated NaHCO 3 was added to terminate the completed reaction. The aqueous layer was extracted with EtOAc (30 mL × 2) after liquid separation, and then the organic layers were combined. The organic layer was washed with saturated brine (40 mL), and then dried with anhydrous MgSO 4 , and the solvent was removed through rotary evaporation under reduced pressure. The raw product was recrystallized with a mixed solvent of petroleum ether and EtOAc, and 6.552 g of white crystals were obtained after filtration and vacuum desiccation.

Preparation of Dihydroartemisinin Amino Derivatives 4a-u
Compound 3, CH 3 CN, K 2 CO 3 and YH were placed in a 100-mL round-bottomed flask, respectively. The mixture reacted at a controlled temperature, while monitoring by TLC. CH 2 Cl 2 (15 mL) and saturated NaCl solution (20 mL) were added. The aqueous layer was extracted after liquid separation with CH 2 Cl 2 (10 mL × 2), and then the organic layers were combined, washed with saturated brine (20 mL) and then dried with anhydrous Na 2 SO 4 ; CH 2 Cl 2 was removed through rotary evaporation under reduced pressure. Pure product was obtained after column chromatography. The experimental results are shown in Table 3.      12β- (2- (1H-1,2,4-Triazol-1-yl) H, m, H-11

Conclusions
In conclusion, molecular docking experiments showed that ART, AS and DHA could dock into AcrB very well, especially DHA and AS; both DHA and AS had same docking pose. The affinity between AS and AcrB seemed weaker than that of DHA, while the succinate tail of AS, like a "bug", could extend in the binding pocket very well. Imitating the parent nucleus of DHA and the succinate tail of AS, twenty-one DHA derivatives 4a-u were designed, synthesized and evaluated. The synergistic activities against E. coli AG100A/pET28a-AcrB showed that the novel compounds 4k, 4l, 4m, 4n, and 4r possessed significant synergism in combination with β-lactam antibiotics although they themselves had no direct antibacterial activity. Among these five DHA derivatives, 4k had the best antibacterial enhancement effect. In conclusion, our results provided a new idea and several candidate antibacterial enhancer compounds against multidrug resistant E. coli.