Quorum Sensing and NF-κB Inhibition of Synthetic Coumaperine Derivatives from Piper nigrum

Bacterial communication, termed Quorum Sensing (QS), is a promising target for virulence attenuation and the treatment of bacterial infections. Infections cause inflammation, a process regulated by a number of cellular factors, including the transcription Nuclear Factor kappa B (NF-κB); this factor is found to be upregulated in many inflammatory diseases, including those induced by bacterial infection. In this study, we tested 32 synthetic derivatives of coumaperine (CP), a known natural compound found in pepper (Piper nigrum), for Quorum Sensing Inhibition (QSI) and NF-κB inhibitory activities. Of the compounds tested, seven were found to have high QSI activity, three inhibited bacterial growth and five inhibited NF-κB. In addition, some of the CP compounds were active in more than one test. For example, compounds CP-286, CP-215 and CP-158 were not cytotoxic, inhibited NF-κB activation and QS but did not show antibacterial activity. CP-154 inhibited QS, decreased NF-κB activation and inhibited bacterial growth. Our results indicate that these synthetic molecules may provide a basis for further development of novel therapeutic agents against bacterial infections.


Introduction
Antibacterial agents are considered to be one of the most important discoveries of the twentieth century. Nevertheless, an increase in the resistance of bacteria to antibacterial agents and a decrease in the development of new antibiotics, according to CDC and WHO reports, are considered a serious worldwide concern [1-3]. To date, conventional antibacterial agents have targeted vital features in the bacteria and have led to selective pressure and the emergence of resistance populations [4]. One feature that seems to be under less selective pressure is the bacterial communication system termed Quorum Sensing (QS). This is a process that regulates the gene expression in response to changes in the population density. The bacteria produce and release small molecules known as Auto-Inducers (AI). When the cell density rises, AI molecules reach a threshold concentration and interact with a receptor protein to form a complex [5]. The AI-receptor complex is a transcriptional regulator protein that complex binds to certain DNA sequences and activates target genes. These create additional AI complexes through a positive feedback [6]. It is well-established that QS regulates numerous virulence genes; thus, these genes can

Synthesis of Monoconjugated Coumaperine Derivatives
The monoconjugated coumaperine derivatives outlined in Figure 2 were synthesized, and different yields were obtained as noted in the figure (47-76%). Detailed synthetic procedures for each individual compound are presented in the Supplementary Data File).

Synthesis of Di-and Triconjugated Coumaperine Derivatives
The di/triconjugated coumaperine derivatives shown in Figure 3 were synthesized, and different yields were obtained, as noted in the figure (12-98%). Detailed synthetic procedures for each individual compound are presented in the Supplementary Data File. The synthesized di-and triconjugated coumaperine derivatives are depicted in Figures 3 and 4. Coumaperine (CP) was acetylated to obtain CP-158 in a moderate (36%) yield. For the present study, piperine was isolated from black pepper [30]. Piperine was deprotected using BBr 3 to obtain CP-209 in a moderate yield (53%). CP-209 was also synthesized by other methods. N-Crotonyl piperidine was condensed with tetrahydropyran-protected 3,4-dihydroxy benzaldehyde in the presence of a base (t-BuOK) to obtain tetrahydropyranprotected CP-209. It was then deprotected with TFA to obtain CP-209 in a moderated yield, 50%. Acetylation of CP-209 with acetic anhydride in the presence of a base (Et 3 N) gave the diacetylated product, CP-281-F1 in a moderate yield (41%). CP-262-F1 and CP-262-F2 were synthesized from the corresponding trimethoxy derivative, CP-27 (shown in Figure 3). CP-27 was demethylated using BBr 3 to obtain monodemthylated derivative, CP-262-F1 and tridemthylated derivative, CP-262-F2 in moderate-to-poor yields (33 and 14%), respectively. The attempt to isolate didemethylated derivatives were not fruitful.  The synthesized compounds were characterized by various spectroscopic techniques, including, FTIR, 1 H-NMR, 13 C-NMR, GC-MS and HRMS/elemental analyses. The spectral data matched with structural attributes for each individual compound are presented in the Supplementary Data File. We previously confirmed the structure of CP-155, CP-209 and CP-262-F2 by single-crystal X-ray diffraction [28].

Antibacterial Effect
We also evaluated all the molecules that showed anti-quorum sensing activity for the antibacterial properties test against both Gram-positive and Gram-negative bacteria. Interestingly, none of the molecules showed antibacterial action against Gram-negative bacteria, and only three of the coumaperine derivatives: CP-9, CP-154 and CP-147, showed antibacterial activity, exclusively against the Gram-positive bacteria. The antibacterial activities of derivatives showing QSI are outlined in Table 2.

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Molecules 2020, 25 CP-102 ++ + * Two tests were carried out using the reporter bacteria: Agrobacterium tumefaciens (KYC55) and Chromobacterium violaceum (CV026). Bacteria were grown overnight in soft agar with the appropriate autoinducer on a Thin Layer chromatography (TLC) plate where the different compounds were dried on 40 mM in ~20-µL acetonitrile, and their degree of QSI was measured semi-quantitatively by color intensity (+ low inhibition, ++ medium inhibition and +++ strong inhibition). Experiments were repeated three times, and the results were determined by considering at least 2 out of 3 experiments with the same results.

Antibacterial Effect
We also evaluated all the molecules that showed anti-quorum sensing activity for the antibacterial properties test against both Gram-positive and Gram-negative bacteria. Interestingly, none of the molecules showed antibacterial action against Gram-negative bacteria, and only three of the coumaperine derivatives: CP-9, CP-154 and CP-147, showed antibacterial activity, exclusively against the Gram-positive bacteria. The antibacterial activities of derivatives showing QSI are outlined in Table 2.
CP-102 ++ + * Two tests were carried out using the reporter bacteria: Agrobacterium tumefaciens (KYC55) and Chromobacterium violaceum (CV026). Bacteria were grown overnight in soft agar with the appropriate autoinducer on a Thin Layer chromatography (TLC) plate where the different compounds were dried on 40 mM in~20-µL acetonitrile, and their degree of QSI was measured semi-quantitatively by color intensity (+ low inhibition, ++ medium inhibition and +++ strong inhibition). Experiments were repeated three times, and the results were determined by considering at least 2 out of 3 experiments with the same results.

Antibacterial Effect
We also evaluated all the molecules that showed anti-quorum sensing activity for the antibacterial properties test against both Gram-positive and Gram-negative bacteria. Interestingly, none of the molecules showed antibacterial action against Gram-negative bacteria, and only three of the coumaperine derivatives: CP-9, CP-154 and CP-147, showed antibacterial activity, exclusively against the Gram-positive bacteria. The antibacterial activities of derivatives showing QSI are outlined in Table 2. Table 2. Antibacterial activity of coumaperine derivatives *.

NF-κB Luciferase Reporter Gene Assay
To further explore the possibility of QS molecules as modulators of NF-κB, we also evaluated the effect of the anti-QS active compounds in reducing NF-κB activation luciferase reporter gene NF-κB inhibition. The compounds that showed a reduction in NF-κB activity were further investigated to determine their potency in a dose response experiment and are presented in Figure 5. At these concentrations, the compounds, except for CP-158, efficiently inhibited NF-κB in L428 cells in a dose-dependent manner. To calculate the median inhibitory concentration (IC 50 ), the percent of NF-κB inhibition was plotted against the log of concentrations. CP-154, IC 50 = 58.8 µM, CP-215, IC 50 = 136.37 µM and CP-286, IC 50 = 45.42 µM. As a positive control, curcumin was used in two concentrations: 80 and 160 µM. Curcumin did not significantly inhibit at 80 µM (NF-κB activation is 91.16% ± 6.5%, p = 0.2). At 160 µM, curcumin significantly inhibited NF-κB (NF-κB activation 19.08% ± 10.7%, p = 0.001).

Quantitation of Activated Nuclear p65-NF-κB Fluorescence
To confirm that the derivatives (CP-154, CP-158, CP-286 and CP-215) inhibit NF-κB, as detected by the luciferase reported gene assay, we used immunostaining. A549 cells were treated with different derivatives, CP-154, CP-158, CP-286 and CP-215 followed by the activation of NF-κB with 2.5 ng/mL of Tumor Necrosis Factor α (TNFα) for 15 min. Fluorescent images were taken from 25 fields in each well in duplicate by the Operetta imaging system and analyzed through the Columbus server. Figure 6A,B are visualizations of the differences between inactive p65 (A) and active p65 followed by TNFα incubation (B). To analyze these results, we defined the active population in the Columbus server ( Figure 6D) and the relative fluorescence values of the nucleus and cytoplasm ( Figure 6C). As shown in Figure 6, CP-154 and CP-215 significantly inhibited NF-κB activated by TNFα, while CP-286 and CP-158 were less active ( Figure 6C). The same results were obtained when the percentage of cells with active NF-κB were scored (Figures 6D and 7).  min, activated p65 in the nucleus. All derivatives were added to the cells at 160 µM for 120 min in DMSO (0.16%) then TNFα at 2.5 ng/mL was added for 15 min. (C) Activated cells (strong nuclear green fluorescence) as a percentage of all the cells in the analyzed field. (D) Ratio of the mean nuclear/cytoplasm fluorescence intensity values in the analyzed fields. Curcumin (CU). Mean ± SD of triplicate samples in two independent experiments. One-way ANOVA comparison of the treatment groups to the control groups DMSO TNF. 95% confidence interval (p-values * ≤0.0332, ** ≤0.0021, *** ≤0.0002). For the comparison between the CU and the CU TNF groups-unpaired two tailed t-test with 95% confidence interval, p-value is 0.0419 (*).

Cell Viability Assay
We tested the cytotoxicity of the compounds that showed QSI on L428 cells as de-

Discussion
Finding molecules that inhibit QS and NF-κB without affecting bacterial growth and preventing drug resistance is of utmost importance. Agents that interfere with bacterial virulence without promoting -resistant, conceivably, will have important therapeutic applications [23]. In the present study, CP, which resembles piperine in structure and is present in low concentrations (<6 ppm) in white pepper, as well as its synthetic derivatives, were evaluated as quorum sensing inhibitors. Although CP is characterized by important pharmacophores such as the Michael acceptor, phenolic and amide moieties, its bioactivity has not been properly explored, probably due to its low bioavailability and long synthetic protocols. Recently, we reported a simple and a robust methodology for the synthesis of CP and its derivatives [28,29]. Following that synthetic process, CP derivatives depicted in Figures 2-4, were synthesized, and characterized for the present study. Initially, the synthesized compounds were evaluated for their QSI properties. These compounds were then examined further for their antibacterial activity, cytotoxicity and NF-κB inhibition activity.
The quorum sensing inhibitory potential of all the CP derivatives was evaluated. CV026 and KYC55 bacteria detect acyl-homoserine-lactone (AHL), a common signal molecule known as autoinducer (AI) of gram-negative bacteria. Different AIs differ in the carbon chain length of the acyl group. CV026 detect N-hexanoyl-L-homoserine lactone (HHL)

Cell Viability Assay
We tested the cytotoxicity of the compounds that showed QSI on L428 cells as de-

Discussion
Finding molecules that inhibit QS and NF-κB without affecting bacterial growth and preventing drug resistance is of utmost importance. Agents that interfere with bacterial virulence without promoting -resistant, conceivably, will have important therapeutic applications [23]. In the present study, CP, which resembles piperine in structure and is present in low concentrations (<6 ppm) in white pepper, as well as its synthetic derivatives, were evaluated as quorum sensing inhibitors. Although CP is characterized by important pharmacophores such as the Michael acceptor, phenolic and amide moieties, its bioactivity has not been properly explored, probably due to its low bioavailability and long synthetic protocols. Recently, we reported a simple and a robust methodology for the synthesis of CP and its derivatives [28,29]. Following that synthetic process, CP derivatives depicted in Figures 2-4, were synthesized, and characterized for the present study. Initially, the synthesized compounds were evaluated for their QSI properties. These compounds were then examined further for their antibacterial activity, cytotoxicity and NF-κB inhibition activity.
The quorum sensing inhibitory potential of all the CP derivatives was evaluated. CV026 and KYC55 bacteria detect acyl-homoserine-lactone (AHL), a common signal molecule known as autoinducer (AI) of gram-negative bacteria. Different AIs differ in the carbon chain length of the acyl group. CV026 detect N-hexanoyl-L-homoserine lactone (HHL) and represent AIs with a shorter carbon chain, whereas KYC55 detect N-3oxooctanoyl-HSL (OOHL) represents AIs with longer carbon chains. Together, these two QS systems are sensitive to a wide range of AIs and serve as sensitive screen models to a wide range of inhibitory molecules [31].
The anti-quorum sensing pattern depicted in Table 1 generally shows that mono conjugated CP derivatives were more effective in comparison to di-and triconjugated derivatives against both bacterial strains, CV026 and KYC55. Thus, shorter conjugation is more effective with most of the derivatives.
Furthermore, monoconjugated derivatives with simple alkyl, phenyl or aryl groups with electron-donating substituents were more effective than monoconjugated derivatives with electron-withdrawing substituents at the aryl group. The diconjugated derivatives with sterically less demanding electron-withdrawing groups, exhibited better anti-quorum sensing activity than the compounds with sterically bulky electron-withdrawing and electron-donating substituents. Similarly, the triconjugated derivatives with sterically less demanding electron-donating substituents were more effective than the derivatives with sterically bulky electron-donating substituents. Thus, monoconjugated derivatives with electron-donating and diconjugated with electron-withdrawing substituents exhibited a high anti-quorum sensing effect against both the bio-reporter bacteria strains, CV026 and KYC55.
The results of the anti-bacterial tests show that CP derivatives that inhibit QS and have the potential to inhibit QS but are not antibiotic, could be used, avoiding the risk of selecting for drug-resistant bacteria. Similar, synthetic derivatives of furanon (a product of marine algae) showed in-vivo QSI activity without antibacterial activity followed by an immune response in the infected animal [32]. Hossain et. al. [33] previously reported that phenolic compounds decrease QS and virulence in PA01 and Chromobacterium violaceum. Thus, there is an increasing number of studies, searching for compounds that target non-vital processes to combat bacteria [34].
Interestingly, the compounds CP-215, CP-154, CP-158 and CP-286 inhibit NF-κB in L428 cells and CP-215, CP-154 and CP-158 inhibit NF-κB in both L428 and A549 cell-lines. In principle, the results obtained here confirmed those obtained using the luciferase reporter gene system [35]. CP-286 and CP-158 were less effective in A549 as compared to their effect in L428 cells. The difference may be due to the compounds' different mechanisms of action, independent or dependent on IκB, since L428 cells lack IkB, while present in A549 cells. Curcumin was used as positive control and significantly inhibit NF-κB activation. The compounds had variable cytotoxicity, CP-215, CP-158 and CP-286 were not cytotoxic. while CP-154 was toxic even at low concentrations.
Thirty-two CP derived molecules were screened for anti QS activity. Twenty three compounds inhibited QS and were further tested for reducing NF-κB activation. Five were found to have both activities (CP-9, CP-154, CP-158, CP215 and CP-286), of which, three were not cytotoxic and did not show antibiotic activity (CP-158, CP-215 and CP-286). These results are summarized in Table 3. CP-9 was previously reported to be cytotoxic and to inhibit NF-κB [29]. Here, we show that CP-9 also has a moderate and low inhibitory effect on QS of CV026 and KYC55, respectively (Table 1) and inhibit bacterial growth of the three gram-positive bacteria were tested (Table 2). These activities suggest that CP-9 and CP-154 may affect multiple targets within bacterial and/or human cells. Interestingly, CP-215, being active but not cytotoxic may show promise as a useful QSI molecule.
* Five compounds with QSI and NF-κB inhibitory properties were tested for antibacterial and cytotoxic activities.
The structure of the derivatives mainly differs in the degree of conjugation and/or in the functional group. Some structural features have an impact on the QSI and NF-κB inhibition activities. For example, CP-273 and CP-154 have a similar structure, containing fluoride and differing only in their carbon chain length. Despite their similarity, they perform very differently in their biological activities. CP-273 has low QSI, no anti NF-κB activity (Table 1), and is not cytotoxic (Supplementary Materials, Table S2). CP-154 on the other hand showed activity but was also cytotoxic (Supplementary Materials, Table  S2). The major differences in their activity might have been determined by the length of their carbon chain. Furthermore, CP-215, PIP and CP-155 differ only in their carbon chain length but show varied biological activity. CP-215 has strong QSI and NF-κB inhibition activities (Table 1, Figures 5-8). By contrast, PIP and CP-155 do not inhibit QS or NF-κB. We also compared CP-270, a mono-conjugate derivative and CP-9, a di-conjugate derivative with no functional group on their aromatic ring. While CP-9 had some antibiotic activity CP-270 do not. CP-270 is highly QSI active in both bio-reporters but CP-9 has moderate and low activity in CV026 and KYC55, respectively (Table 1). In this study, CP-270 had no anti-inflammatory activity, while CP-9 demonstrated strong NF-κB inhibition [29]. The unique contribution of the functional group can be observed in the couple CP-38 and CP-147, both di-conjugates, where the differences in their structure area thiomethyl functional group (-SCH 3 ) and in CP-38 a methoxy (-OCH 3 ) moiety. This relatively small change has a significant impact on their functions, CP-147 inhibits Staphylococcus aureus growth (Table 2), has a low QS inhibition effect on CV026, does not affect QS in KYC55 (Table 1) and has no effect on NF-κB. The activities of CP-38 were listed above (and summarized in Figure 8). The different functional group among them is interesting, where oxygen is more active than thio at this position.
Here, we show that synthetic derivatives of natural compounds often have improved therapeutic values over the parental molecule, as they enable us to solve problems of solubility, toxicity, activity and specificity. In conclusion, QS inhibition is a promising approach to prevent bacterial virulence, focused on the disturbance of bacterial communication. Coupled with the ability to inhibit NF-κB, we believe of our derivatives are suitable candidates for further development as therapeutic substances to prevent complications resulting from bacterial infections. Samples were made as pellet with KBr and recorded. High-resolution mass spectra were recorded on Electrospray Ionization mode on Agilent 6520 (Q-TOF) (Santa Clara, CA, USA) mass spectrometer in positive (ESI + ) ion mode. Mass spectra were recorded on Perkin Elmer Clarus 600/Shimadzu QP2020 GC-MS spectrometer (Kyoto, Japan) in EI mode. Melting points were recorded with REMI DDMS 2545 (Cama Industrial Estate, Goregaon (East), Mumbai, India). The instrument is calibrated with benzoic acid before the measurement.

Synthesis Methods
The mono-, di-and triconjugated coumaperine derivatives were synthesized as described in detail in the Supplementary Results Section. In short, coumaperine derivatives with one alkene bond between the aromatic ring and piperidine are termed monoconjugated coumaperine derivatives and those with two and three alkene bonds are termed diand triconjugated coumaperine derivatives respectively (Figure 9). The monoconjugated coumaperine derivatives were synthesized in two steps [28,29]. Knoevenagel condensation of aldehyde and malonic acid in presence of base yields cinnamic acid derivatives. It was then converted to the corresponding acid chloride and subsequently reacted with piperidine allowing us to obtain monoconjugated coumaperine derivatives (refer to ESI for detailed synthetic procedure). CP-215 was demethylated to obtain dihydroxy derivative, CP-237. Demethylation was achieved with AlCl 3 at room temperature ( Figure 10). Di-and triconjugated derivatives were prepared from the corresponding aldehyde and crotonic/sorbic acid according to our synthetic procedure reported earlier [28,29]. Crotonic/sorbic acid was converted to acid chloride by reacting with thionyl chloride and subsequently reacted with piperidine to obtain crotonyl/sorbyl piperidine. It was then condensed with aromatic aldehydes in presence of base (KOH/t-BuOK) at room temperature to obtain the final product, di/triconjugated coumaperine derivatives (Figure 3 AT media:50 mL × 20 AT-buffer, 50 mL × 20 AT salts, 10 mL 50% (w/v) glucose and add 890 mL to a final volume of 1 L). For O.N. at 30 • C in an orbital shaker, into preheated water agar with 25 mL AT buffer and 60 µg/mL Xgal and 5-nM 3-oxo-c8 (O1764, Sigma-Aldrich, Saint louis, MO, USA), 25 mL of the starter was added. Then, that mixture was loaded on the TLC in a glass plate for O.N. incubation in 30 • C. The next day, the inhibition zone was measured. The bacteria produce blue pigment during QS and when QS is inhibited no pigment is produced. KYC55 (pJZ372) (pJZ384) (pJZ410) preservation in tetracycline (1 µg/mL), spectinomycin (100 µg/mL) and gentamicin (100 µg/mL) (all antibiotics from Sigma Aldrich, Saint louis, MO, USA) in AT media O.N. then centrifuged and suspend in 15% glycerol frozen and maintained at −80 • C [31,36].

Diffusion Discs Test
All derivatives were dissolved in acetonitrile to a final concentration of 40 mM. Twenty microliters were loaded onto Whatman filter paper discs and dried. Bacillus subtilis, Staphylococcus aureus and Acinetobacter baumannii were grown in LB (Lenox) O.N. One hundred microliters. from the starter culture was plated on LB agar (Difco Luria-Bertani medium, Le Pont se Claix, France) dishes. Streptococcus sobrinus and Streptococcus mutans grown in Brain Heary Infusion (BHI) (Himedia, MS, India) O.N. were similarly plated on BHI agar dishes. Discs were laid and incubated O.N. in 37 • C and the diameter of inhibition of bacterial growth was measured.

NF-κB-Luciferase Reporter Gene Assay
L428 cells stable transfectants with the luciferase NF-κB-Luc reporter gene were generated, as described in Ozer et al. 2009 [35] and were maintained in 500 µg/mL of the G418 (G418 disulfate salt, Sigma-Aldrich, Saint louis, MO, USA). The cells (5 × 10 5 /well in tripli-measured at 450 nm and 650 nm in an ELISA plate reader (Multiskan Spectrum, Version 1.2. ThermoFisher scientific, Waltham, MA, USA) were recorded. Absorption values at 650 nm were subtracted from the blank and the 450 nm values. The results were normalized to the control (treated with DMSO). To determine the LC 50 values of the derivatives, the Probit transformation was done only on samples with R 2 values > 0.85 and only on derivatives that showed significant cytotoxicity.

Statistical Analysis
Two-way ANOVA followed by Tukey's multiple comparisons test or one-way ANOVA was performed using GraphPad Prism version 8.0.1 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com).