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Discovery and preliminary SAR of 14-aryloxy-andrographolide derivatives as antibacterial agents with immunosuppressant activity

Antibacterials (which restore gut flora balance) and immunosuppressants (which correct immune defects) are two important and effective therapeutic agents for the treatment of inflammatory bowel disease (IBD) in clinical use today. Since the structural skeleton of andrographolide, isolated from Andrographis paniculata, has become known as a natural antibiotic with anti-inflammation and heat-clearing and detoxifying properties, 14-aryloxy andrographolide derivatives have been designed, synthesized, and tested for their antibacterial effects on E. coli, S. aureus, and E. faecalis, which are related to IBD. It has been discovered in this study that the andrographolide skeleton is more selective against E. faecalis, the 14-aryloxy group with basicity is important for antibacterial functions, and the 14-(8′-quinolinyloxy) group is a good pharmacophore with antibacterial activity. In addition, we found that 7b1 and 8b1 are good and selective inhibitors of E. faecalis; two 14β-(8′-quinolinyloxy) andrographolide derivatives, 6b17 and 9b, exhibit good activity against E. coli, S. aureus, and E. faecalis. Likewise and importantly, further exploration of immunosuppressant activity for IBD shows that compound 7b1 is a selective inhibitor of the TNF-α/NF-κB signaling pathway, whereas 8b1 is selectively active against the TLR4/NF-κB signaling pathway; moreover, the compounds 6b17 and 9b are active in inhibiting the IL-6/STAT3, TLR4/NF-κB, and TNF-α/NF-κB signaling pathways. Based on these results, we have further focused on the development of dual function inhibitors of IBD as antibacterial and immunosuppressant agents by structural modification of andrographolide.


Introduction
Inammatory bowel disease (IBD) 1 is an autoimmune disease that is characterized by relapsing and remitting chronic inammation of the gastrointestinal tract; it is believed to be caused by unbalanced host-commensal microbiota and a common immune defect in addition to genetic predisposition. 2 Immune responses have been recognized to be related to IBD pathogenesis, 3 and a recent study demonstrated that the innate immune system is a major determinant of the serum and tissue proles of IBD, 4 further explaining the existence of regulatory cytokines that are implicated in immune responses related to IBD. 5 Especially, overexpression of proinammatory cytokines, such as interleukin-6 (IL-6), toll-like receptor 4 (TLR4), and tumor necrosis factor-a (TNF-a), is crucial for IBD onset and progression; these proinammatory cytokines, as inammatory mediators, are connected with the activation of nuclear factor of k light polypeptide gene enhancer in B-cells (NF-kB) and signal transducer and activator of transcription 3 (STAT3). 5 As NF-kB and STAT3 are known to play central roles in regulating inammatory responses in patients with IBD, they are recognized as important targets for therapeutic intervention of IBD. 6 Gastrointestinal microbiota are believed to be commensal and mutualistic to humans and animals and to have health benets for humans and animals in some aspects. 7 Signicant imbalances of gastrointestinal ora are observed in patients with IBD as compared to the case of healthy people; for example, proteobacteria and actinobacteria appear to dominate in people with ulcerative colitis (UC), whereas Enterococcus (E.) faecium and several proteobacteria over-inhabit the gastrointestinal tracts of people with Crohn's disease (CD). 8 Although the etiology and pathogenesis of IBD are not fully understood, E. coli and E. faecalis are recognized as pathogens of IBD. 9 It is controversial whether S. aureus infection is involved in early lesions or established lesions of IBD; however, S. aureus infection occasionally may occur and complicate IBD during the course of the disease. 10 Metabolites from certain members of the gut ora may have causal contributions to disorders such as obesity and colon cancer by changing host signaling pathways. 11 In addition, the intrusion of gut ora components into other host compartments can lead to sepsis. 11 Therefore, immunosuppressants (which correct immune defects) and antibacterials (which restore ora balance) are two important and effective therapeutic agents to treat IBD in clinical use. Considering this, we envisage that it is a meaningful exploration to discover single compounds with dual functions of antibacterial and immunosuppressant activities.
Andrographis paniculata [Burm. F.] Nees, an herb known as a "natural antibiotic", is commonly used in China, India and Southeast Asia for the treatment of a large variety of illnesses, especially infectious diseases, by reducing inammation and "heat-clearing and detoxifying". Andrographolide (1, Scheme 1), a bicyclic diterpenoid lactone, is a major active component isolated from Andrographis paniculata [Burm. F.] Nees; 12 its derivatives, 13 such as "Chuanhuning", 14,15 have been used in China to treat bacterial and viral infections for many years. Numerous andrographolide derivatives have been designed and synthesized in recent years. 16 The antibacterial activities of andrographolide and its derivatives are not potent and show only minimal or marginal direct inhibition of bacterial growth; however, it was discovered that andrographolide inhibits the quorum sensing (QS) system 17 of P. aeruginosa and that andrographolide derivatives block biolm formation 18 of P. aeruginosa, indicating that andrographolide and its derivatives can directly inhibit bacterial growth or/and infection by specic mechanisms and modes of action. On the other hand, some evidence has been found that the anti-inammatory effects of andrographolide are related to regulation of the immune system 19 and that andrographolide suppresses TLR4 expression and NF-kB signaling in multiple myeloma cells. 20 Multitargeting andrographolide and its analogs have potential use in the prevention and treatment of metabolic syndrome 21 and stroke 22 via the NF-kB signaling pathway. The andrographolide derivative isoandrographolide signicantly inhibited the release of NO and prostaglandin E2 and the production of interleukin-1b (IL-1b) and IL-6 in lipopolysaccharide (LPS)stimulated J774A.1 macrophage cells in a dose-dependent manner. 23 Andrographolide analog AL-1 improved insulin resistance by down-regulating the NF-kB signaling pathway. 24 Our previous report 25 revealed that some analogs of andrographolide play a role in the attenuation of innate immunity; these were identied as potential immunomodulatory inhibitors of TLR signaling with distinct regulation of NF-kB family members. Furthermore, one compound effectively reduced LPSinduced pulmonary injury in a mouse in vivo study; biochemical results showed that the nucleus translocation of phosphorylated p65 and serum pro-inammatory cytokines decreased. Based upon these facts, we are interested in developing dualfunction analogs of andrographolide as immunosuppressants to inhibit excessive immunity to "self" gastrointestinal microbiota and also as antibacterial agents to control infection by specic "commensal/mutualistic" gut ora; such compounds should be valuable in the treatment of IBD.
In our search for dual-functional 14-aryloxy andrographolide analogs inhibiting both bacterial growth and innate immunity, we launched a panel of screening platforms of these analogs to study their antibacterial activities against E. coli, S. aureus and E. faecalis and preliminary structure-activity relationship (SAR) studies as the rst step. Selected active anti-bacterial compounds were explored as immunosuppressants to protect the host from abnormal immune response initiated by intestinal commensal/mutualistic bacteria. In this paper, we describe the synthesis and evaluation of a series of 14-aryloxy andrographolide compounds against E. coli, S. aureus and E. faecalis as well as against the IL-6/STAT3, TLR4/NF-kB and TNFa/NF-kB signaling pathways of innate immune response and their preliminary SAR. It was discovered that analogs of andrographolide are more sensitive to E. faecalis and that active antibacterial compounds are greatly superior to andrographolide to attenuate innate immune response. Our results revealed that the andrographolide skeleton has immunosuppressant and antibacterial properties and that these analogs of andrographolide can potentially be used to treat IBD.

Antibacterial activity and SAR analysis
Numerous bacteriostatic and bactericidal agents have been discovered and developed against bacterial growth and infection; however, bacterial resistance to currently used antibacterial drugs is becoming a major problem in clinical practice, especially hospital-acquired infections by these drug-resistant bacteria. 27 To meet the demand of clinical medication for the treatment of digestive diseases caused by gastrointestinal bacterial infection, antibacterial drugs with diverse and novel structural origins are required. 28 On the basis of reported literature studies stating that andrographolide and some of its derivatives may function as antibiotics, we are pursuing the discovery of more potent and specic andrographolide derivatives for the development of chemotherapeutical agents against bacterial infection, especially for inhibiting gastrointestinal bacterial infection in the development of IBD. The strategy in this study was to submit these synthesized compounds to a preliminary screening test against E. coli, S. aureus and E. faecalis (ciprooxacin was used as a reference compound; see Table 1). Because these andrographolide analogs did not obviously function as bactericidal agents (data not shown), bacteriostatic screening was employed with gradient concentrations and the percent growth was plotted versus test concentration to afford the IC 50 values. 29 The current antibacterial activity results are listed in Table 1.
Based on the above data, further exploration to introduce more complex aryl groups at the 14-position were conducted. 14b-(1 0 -Naphthyloxy) compound 5b14 exhibited very weak inhibitory activity only to E. faecalis, and 14b-(1 0 -naphthyloxy) diol compound 6b14 expressed very weak activity against all three bacteria. Interestingly, the introduction of a 2 0 -nitropyridinyl-3-oxy group at the 14-position increased the activity of all four derivatives 5a15, 5b15, 6a15 and 6b15 against E. faecalis; 14a-3,19-acetonylidene analog 5a15 was the most active, while 14a-3,19-diol analog 6a15 was the least active. Moreover, 5b15 was more active than its corresponding 2 0 -nitro phenoxy derivative 5b6, but 6b15 was less active than 2 0 -nitro phenoxy analog 6b6. Corresponding to the replacement of phenoxy groups with pyridinyloxy groups, substitution of naphthyloxy by quinolinyloxy was subsequently studied. Unlike 14b-(1 0 -naphthyloxy) analogs 5b14 and 6b14, four analogs of 14-(4 0 -quinolinyloxy) derivatives 5a16 (clog P 6.8988), 5b16 (clog P 6.8988), 6a16 (clog P 4.8578) and 6b16 (clog P 4.8578) expressed antibacterial activity against E. faecalis; however, none of these was active against E. coli, and two 14a-isomers, 5a16 and 6a16, were very weak inhibitors of S. aureus. The clog P values of 3,19protected compounds 5a16 and 5b16 were higher than 5.0 and higher than those of 6a16 and 6b16; 5a16 and 5b16 were more active against E. faecalis than the corresponding 3,19-diol compounds 6a16 and 6b16, respectively. Unlike the activity relationships between 5a15, 5b15, 6a15 and 6b15, the activity variation patterns against E. faecalis of 5a16 and 5b16 were parallel to those of 6a16 and 6b16 and 14b-isomers 5a16 and 6a16 were slightly more active than 14a-isomers 5b16 and 6b16, respectively. These data imply that a positive charge of nitrogen in the 14-aryloxy group under physiological conditions may play a role in antibacterial activity for the substitutions of phenoxy groups by pyridinyloxy groups and of naphthyloxy groups by quinolinyloxy groups.
Continued exploration in the quinolinyloxy line disclosed that 14-(8 0 -quinolinyloxy) derivatives exhibited novel antibacterial activity proles. In contrast to the 14-(4 0 -quinolinyloxy) derivatives, the 14-(8 0 -quinolinyloxy) derivatives were active against all three bacteria; they also showed more selective inhibition of E. faecalis than of E. coli or S. aureus, similar to the other series of 14-aryloxy andrographolide derivatives in this work. Specically, 14a-3,19-acetonylidene compound 5a17 (clog P 6.77355) was more active against E. faecalis than 14a-3,19-diol compound 6a17 (clog P 4.73255), while 14b-3,19-acetonylidene compound 5b17 (clog P 6.77355) was less active against E. faecalis than 14b-3,19-diol compound 6b17 (clog P 4.73255); this activity pattern against E. faecalis is the same as that of 14-(2 0 -nitro-pyridinyl-3-oxy) derivatives 5a15, 5b15, 6a15 and 6b15. Importantly, 6b17 expressed good antibacterial activity against all three bacteria (Table 1) and was the most potent inhibitor of E. faecalis in this paper (Table 1). These data illustrate that the 14-(8 0 -quinolinyloxy) compounds showed superior results against all three bacteria compared to their corresponding 14-(4 0 -quinolinyloxy) compounds. 19-Acetylated 7b1 (clog P 5.64055) and its 3-ketone 8b1 (clog P 5.37725) had almost no inhibitory activity toward E. coli and S. aureus but were still very active against E. faecalis. However, 19-silylated 7b2 (clog P 8.10955) had decreased inhibitory activity toward all three bacteria and possessed only mild activity against E. faecalis, while the antibacterial activity of 3-ketone 8b2 (clog P 7.85675) was very weak against all three bacteria. It is interesting that 3-keto-19-alcohol compound 9b (clog P 4.47495) regained the same antibacterial activity against all three bacteria (Table 1) as 6b17, although its inhibitory activity toward E. faecalis was slightly less potent than that of 6b17. By combining these data, it is suggested that in addition to the importance of the andrographolide skeleton, the 14-(8 0 -quinolinyloxy) group is essential to antibacterial activity; moreover, modications at the 3-or/and 19-positions are not ignorable, and 19-acetylation greatly reduced inhibitory activity to E. coli and S. aureus while transformation of 3-alcohols into 3-ketones slightly inuenced the antibacterial activity.

Inhibition of innate immune response and SAR analysis
Considering that initiation of a dysregulated immune response within the intestinal mucosa by "self" gut ora and/or food is due to immune defects in IBD, reduction of over-immunity is an effective treatment of IBD. 3,30 TNF-a, the best studied NF-kB activator, is correlated with transformation of NF-kB by various stimuli from its inactive form to its active form. TLRs are involved in the regulation of innate and adaptive immunity, 31 and LPS-activated TLR4 regulates the expression and nucleus translocation of NF-kB. 32 IL-6 is a crucial pathogenic mediator in IBD that functions by triggering cellular effects and functions via STAT3 signaling. The proinammatory IL-6/STAT3dependent biological network is upregulated in active IBD patients and is considered to be an important pathogenic factor in IBD onset 33 and progression. 34 Based on these facts, immunosuppressants, corticosteroids and anti-TNF-a antibodies have become commonly used drugs in IBD treatment; they may affect IBD progression by interfering with cellular oxidative stress and cytokine production. 35 We utilized luciferase reporters bearing the promoter regions of TNF-a/NF-kB, TLR4/NF-kB, and IL-6/STAT3 (ref. 25) to examine the activities of the antibacterial active andrographolide analogs in the regulation of signaling pathways that govern inammatory response (Table 2). It was noted that these derivatives did not show obvious cytotoxicity to AD-293 cells; DCB-3503 was used as a positive compound 36 (Table 2). Our previous study revealed that compound 6b3 is active against the TNF-a/NF-kB and TLR4/NF-kB signaling pathways; 25 however, 6b3 does not have antibacterial activity (Table 1). 14a-Compounds 5a17 and 6a17 are active against the IL-6/STAT3 signaling pathway but do not inhibit the NF-kB signaling pathways. 14b-Compounds 5b17 and 6b17 are active to suppress the three signaling pathways (Table 2), suggesting that 3,19-acetonylidene does not obviously affect the inhibition of these signaling pathways; although the feature of 3,19-acetonylidene may contribute to the distinct activities of 5b17 and 6b17 against E. faecalis, 6b17 is also 3 times more active than 5b17 against E. faecalis. Compounds 7b1 and 8b1 possess similar inhibitory activities to E. faecalis; however, 3-alcohol-19acetylated compound 7b1 shows selective anti-TNF-a/NF-kB activity, while 3-keto-19-acetylated compound 8b1 is selectively active against the TLR4/NF-kB signaling pathway. Like their similarity in inhibitory activity against E. faecalis, E. coli and S. aureus (Table 1), 3-keto-19-alcohol compound 9b1 suppresses three signaling pathways in the same fashion as 6b17 (Table 2). These interesting data suggest that dual-functional analogs of andrographolide can behave as antibacterial and immunosuppressant agents and possibly have synergistic effects in IBD treatment.

Conclusion
Bacterial infections have long been regarded as a formidable enemy of mankind. Bacterial pathogens dramatically affect the normal function of host cells, including immune response, for their own benet during infection; eventually, they destroy or damage host cells. As reconstitution of gut ora balance and correction of immune defects are two important strategies for IBD treatment, we attempted to discover dual-functional andrographolide analogs for use as antibacterial and immunosuppressant agents that could synergize ultimate efficacy.
In this study, 14-aryloxy andrographolide derivatives were designed, synthesized and tested against the growth of E. coli, S. aureus and E. faecalis. Preliminary data suggest that the andrographolide skeleton is primarily more selective against E. faecalis; modications at the 3-or/and 19-positions are not ignorable; the 14-aryloxy moiety strongly determines essential efficacy against bacteria growth; 14a-and 14b-isomers exhibit different antibacterial activities; and the substitution positions at the phenoxy groups and the structural features of the substitution groups may alter the inhibitory activities of the compounds. The current data imply that nitrogen-containing 14-aryloxy arenes may play an enhanced role in antibacterial activity upon the substitution of phenoxy groups by pyridinyloxy groups and of naphthyloxy groups by quinolinyloxy groups, suggesting the possibility that the nitrogen of the arene group is charged under physiological conditions. Moreover, it was revealed that the 14-(8 0 -quinolinyloxy) group is an important pharmacophore for antibacterial activity and that 14b-(8 0 -quinolinyloxy) andrographolide derivatives exhibit superb inhibitory activity toward E. faecalis. Importantly, andrographolide analogs with antibacterial activities also possess immunosuppressant activities against the IL-6/STAT3, TLR4/NF-kB and/or TNF-a/NF-kB signaling pathways, enabling us to conclude that dual functional andrographolide analogs can play synergistic roles in IBD treatment.
The results of this study suggest that the development of andrographloide analogs as antibacterial and immunosuppressant agents is possible. Further discovery of more potent structural cores, structural tuning of the 14-aryloxy substitutions and modications at other positions are underway on the basis of these ndings.

Materials and methods
4.1. General information for chemistry 1 H and 13 C NMR spectra were recorded on a Bruker AV-400 spectrometer at 400 and 100 MHz, respectively, in CD 3 Cl, CD 3 OD, (CD 3 ) 2 SO and C 6 D 6 as indicated. Coupling constants (J) are expressed in hertz (Hz). Chemical shis (d) of NMR are reported in parts per million (ppm) units relative to the solvent. High resolution ESI-MS spectra were recorded on an Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer. Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purication. Melting points were measured using a YRT-3 melting point apparatus (Shanghai, China) and were uncorrected.

. Preparation of compounds 6a and 6b
General method for synthesis of 6a and 6b. At room temperature, 1.0 mmol 5a or 5b were dissolved in 15 mL methanol, and 0.1 mmol TsOH$H 2 O was added. The reaction was stirred at rt and was generally complete in 5 min. Aer the reaction was complete, the reaction mixture was treated with sol. sat. NaHCO 3 and extracted with ethyl acetate. The organic phase was washed with brine and dried over anhydrous Na 2 SO 4 . The solvent was removed in vacuo, and the residue was further puried using ash column chromatography eluting with petroleum ether and ethyl acetate to afford 6a or 6b.
(14a)-(2 0 -Carboxy ethyl ester-4 0 -nitro)phenoxy-andrographolide (6a1    50 ) against signaling pathways and cytotoxicity (CC 50 ) of testing compounds in AD-293 cells. Cells from the AD-293 cell line, a derivative of the HEK293 cell line with improved adherence and plaque formation properties, were purchased from Stratagene (La Jolla, CA, USA). The protocols were adopted and modied from our previous report. 25 All compounds were dissolved in DMSO at 10 mM as a stock solution. The nal concentration of DMSO was 0.1% in the culture medium. AD-293 cells bearing luciferase reporters with promoter regions of IL-6/STAT3 and TNF-a/NF-kB in pGL4.20 vector (Promega) were maintained in DMEM high glucose medium supplemented with 10% FBS and 1% penicillin streptomycin in the presence of 1 mg mL À1 puromycin. AD-293 cells overexpressing TLR4 and stably transfected with NF-kB reporter were maintained in DMEM high glucose medium in the presence of 1 mg mL À1 puromycin and 10 mg mL À1 blasticidin. All cells were maintained in a humidied incubator at 37 C in 95% air and 5% CO 2 .
Cytotoxicity of testing compounds. AD-293 cells were plated in a 96-well plate at a concentration of 1.0 Â 10 5 cells per well overnight to allow cell attachment. Working solutions of the tested compounds, the positive drug, DCB-3503 36 of a cryptopleurine analog, or the DMSO vehicle as a control were dispensed appropriately into the partitioned 96-well plates, which were then incubated for another 24 h. Then, the medium was discarded and the cells were incubated for 4 h at 37 C in MTT solution (nal concentration 0.5 mg mL À1 ). The solution was then replaced with 100 mL DMSO to dissolve the violet formazan crystals in the intact cells. Cell growth was assessed by MTT according to the manufacturer's protocol. The absorbance was measured at 570 nm as the reference wavelength. The cytotoxicity as the CC 50 value (concentration of 50% cell growth/ viability inhibition) was calculated based on the percentage of cell viability data compared to the control group. Each concentration was repeated 3 times independently.
Signaling pathway reporter assay. Reporter cells were treated with 50 ng mL À1 TNF-a to stimulate the NF-kB signaling pathway, 1 mg mL À1 LPS to stimulate the TLR4/NF-kB signaling pathway, and 2.5 mg mL À1 IL-6 to stimulate the IL-6/STAT3 signaling pathway. The medium was removed at the end of the treatment, cell extracts were prepared, and the luciferase activity was measured using a Luciferase assay kit (Promega) according to the manufacturer's instructions. EC 50 was dened as the concentration of drug that inhibited stimulatortriggered luciferase reporter activation by 50% aer continuous drug exposure for 4 (TNF-a/NF-kB) or 16 (IL-6/STAT3 and TLR4/ NF-kB) hours. Each concentration was repeated 3 times independently.

Author contributions
GCZ, DW and YW conceived and supervised the project, analyzed the data and wrote the paper. GCZ, FL, DS, XN, ZL and DW designed the andrographolide derivatives; FL, DS, XN, ZL and DW conducted the syntheses of andrographolide derivatives. YW, XML, SRC, QZ, and YTW designed the biological experiments. XML, SRC and QZ conducted the biology experiments. All authors have read and approved the nal manuscript.

Conflicts of interest
There are no conicts to declare.