Quorum sensing and nf-κb inhibition of synthetic coumaperine derivatives from piper nigrum

Article abstract of DOI:10.3390/molecules26082293

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.

Full text of DOI:10.3390/molecules26082293

molecules
Article
Quorum Sensing and NF-κB Inhibition of Synthetic
Coumaperine Derivatives from Piper nigrum
Yael Kadosh 1,†, Subramani Muthuraman 2,†, Karin Yaniv 1 , Yifat Baruch 1, Jacob Gopas 3,4
,
Ariel Kushmaro 1,
*
and Rajendran Saravana Kumar 2,
*
1
Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben Gurion University of the
Negev, Be’er Sheva 8410501, Israel; shlicter@post.bgu.ac.il (Y.K.); kariny@post.bgu.ac.il (K.Y.);
yifatbar@post.bgu.ac.il (Y.B.)
2
3
4
Chemistry Division, School of Advanced Sciences, Vellore Institute of Technology, Chennai 600127, India;
muthu.ramans2014@vit.ac.in
Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben Gurion University
of the Negev, Be’er Sheva 8410501, Israel; jacob@bgu.ac.il
Department of Oncology, Soroka University Medical Center, Ben Gurion University of the Negev,
Be’er Sheva 8410501, Israel
*
†
Correspondence: arielkus@bgu.ac.il (A.K.); sar.org@gmail.com (R.S.K.)
These authors contributed equally to this work.
Abstract: 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
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Kadosh, Y.; Muthuraman,
S.; Yaniv, K.; Baruch, Y.; Gopas, J.;
Kushmaro, A.; Kumar, R.S. Quorum
Sensing and NF-κB Inhibition of
Synthetic Coumaperine Derivatives
from Piper nigrum. Molecules 2021, 26,
2293. https://doi.org/10.3390/
molecules26082293
natural compound found in pepper (Piper nigrum), for Quorum Sensing Inhibition (QSI) and NF-
inhibitory activities. Of the compounds tested, seven were found to have high QSI activity, three
inhibited bacterial growth and five inhibited NF- B. 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- B activation and QS but did not show antibacterial activity. CP-154 inhibited QS,
decreased NF- B activation and inhibited bacterial growth. Our results indicate that these synthetic
κB
κ
κ
κ
molecules may provide a basis for further development of novel therapeutic agents against bacterial
infections.
Academic Editors: Raffaele Capasso
and Lorenzo Di Cesare Mannelli
Keywords: quorum sensing; antibacterial; NF-kB; Piper nigrum; coumaperine; plant natural-based
compounds; amide alkaloids
Received: 10 March 2021
Accepted: 11 April 2021
Published: 15 April 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
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 [
antibacterial agents have targeted vital features in the bacteria and have led to selective
pressure and the emergence of resistance populations [ ]. One feature that seems to
1–3]. To date, conventional
4
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
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
and interact with a receptor protein to form a 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 [ ].
It is well-established that QS regulates numerous virulence genes; thus, these genes can
5]. The AI-receptor complex
6
Molecules 2021, 26, 2293. https://doi.org/10.3390/molecules26082293
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2293
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be used as possible targets for attenuation and as a treatment for bacterial infection [7–12].
The main advantages of such QS inhibitors are the avoidance of rapid selective pressures
that conventional antibiotics generate and, thus, may provide a possible treatment for
existing infections by multi-drug resistance bacteria. In mammals, bacterial infections alter
the immune system and cause an inflammatory reaction that can by itself be an issue.
Such an inflammation reaction is a stress response that includes a number of signals
(e.g., cytokines, chemokines, biogenic amines and eicosanoids) that induce changes in
diverse biological processes, suppressing the local or systemic homeostasis. The immune
responses to infection are tightly regulated inflammation balancing between elimination
of the intruder and the possible damage to the tissues [13,14]. Aberrant regulation of the
immune responses to an infection may cause an increase in tissue damage, ultimately
leading to increased mortality [15]. Therefore, immune modulation may provide an
important method of treatment for infection. A promising target for immune modulation
is the Nuclear Factor kappa B (NF-
involved in inflammation, cell proliferation and apoptosis. The NF-
five members: RelA (p65), relB, c-Rel, p105/p50 and p100/p52 [16]. Moreover, NF-
κ
B) transcription factor that regulates over 500 genes
κ
B family comprises
κ
B
displays an important role in first-line protection against intruders (e.g., viruses or bacteria)
and is evolutionary conserved [17]. Indeed, some pathogens evolved to modulate this
transcription system in order to avoid or to take advantage of the immune responses [18,19].
Examples for this is what occurs during long-term infections of Pseudomonas aeruginosa in
cystic fibrosis patients, as well as what occurs during urinary tract infections. P. aeruginosa
infections were found to be able to avoid phagocytes and leukocytes and cause tissue
damage and apoptosis with rhamnolipids and exotoxins [20]. The QS signal molecule
of P. aeruginosa 3-oxo-C12-HSL was found to inhibit the degradation of I
NF- B, interestingly, which enables a longer activation of NF- B, which is likely to promote
the persistence of the infection [21]. In an additional example: Acinetobacter baumannii
pneumonia causes systematic inflammation through the activation of NF- B, increasing the
κB, inhibitor of
κ
κ
κ
production of proinflammatory cytokines and increased expression of Toll-Like Receptor 4
(TLR4). Interestingly, treatment with the anti-inflammatory cytokine Interleukin 33 (IL-33)
depresses the inflammation storm and decreases the mortality in a model of A. baumannii-
infected mice [22]. Finding a molecule that can inhibit both QS and NF-
κ
B may therefore
present superior therapeutic value against bacterial infections.
In recent years, plant-derived natural products known to contain NF-
κ
B modulatory
activity were investigated. Additionally, natural compounds from plants that have long
been used to treat microbial infections have gained much attention as a source of quorum
sensing inhibitors [23]. Malabaricone C isolated from the bark of Myristica cinnamomea
inhibited quorum sensing–regulated biofilm formation in P. aeruginosa (PAO1) and violacein
production by Chromobacterium violaceum CV026 [24]. Recently, dietary phytochemicals
with medical uses in humans have been investigated for anti-quorum sensing activity. In
addition, many plant-derived natural products are known to contain NF-
κB modulatory
activity, such as curcumin, which inhibits the phosphorylation of I and p65 [16]. The
κBα
plant-derived curcumin is a major constituent of turmeric that was found to inhibit quorum
sensing-regulated biofilm formation in uropathogens [25]. Piperine, a bioactive constituent
of black pepper, was also shown to inhibit biofilm formation by interfering with quorum
sensing activity in Streptococcus mutans [26]. (Figure 1). An additional natural product,
Coumaperine, an amide alkaloid found in white pepper (Piper Nigrum), structurally similar
to Piperine whose bioactivity has also been explored [27]. Recently, our group reported
the 5-LOX inhibitory activity [28] and antiproliferative activity of coumaperine derivatives
against two cancer cell lines, L428 and A549, and the inhibition of A549 cell migration [29].
However, the anti-QS activity of coumaperine and its derivatives are still unexplored.
Therefore, in the present study, we tested 32 derivatives of coumaperine to determine their
quorum sensing inhibition (QSI), antibacterial and NF-κB inhibitory activities.

Molecules 2021, 26, 2293
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Figure 1. Few examples of natural product-based quorum sensing inhibitors.
2. Results
2.1. 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).
Figure 2. Synthesis of monoconjugated coumaperine derivatives.
2.2. 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 pro-
cedures 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 BBr3 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 tetrahydropyran-
protected 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 (Et3N) 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 BBr3 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.

Molecules 2021, 26, 2293
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Figure 3. Di and triconjugated coumaperine derivatives synthesized.
Figure 4. Diconjugated coumaperine derivatives synthesized.
The synthesized compounds were characterized by various spectroscopic techniques,
including, FTIR, 1H-NMR, 13C-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].
2.3. Quorum Sensing Inhibition Assay
The effects of all the synthesized compounds evaluated for quorum sensing inhibition
against two bioreporter bacteria strains, Chromobacterium violaceum (CV026) and Agrobac-
terium tumefaciens (KYC55) are summarized in Table 1. The monoconjugated derivatives
with a simple phenyl group (CP-270), methylenedioxy-substituted (CP-215), and alkyl-
substituted compounds (CP-296), were highly active in both tests (Table 1, entry 1–3). The
monomethoxy derivative, CP-282 (Table 1, entry 4) was more effective against CV026 than
KYC55, while the dihydroxy derivative CP-289 (Table 1, entry 5) was more effective against
KYC55 than CV026. The dihydroxy compound CP-237 and cycloalkyl derivative CP-295
(Table 1, entries 6 and 7) displayed moderate activity against both the bacterial systems. The
monoconjugated derivative with an electron-withdrawing substituent nitro group (CP-286)
exhibited low and moderate activity against CV026 and KYC55, respectively (Table 1, entry

Molecules 2021, 26, 2293
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8). CP-273 exhibited low activity and was inactive against CV026 and KYC55, respectively.
CP-291 was inactive against CV026 and exhibited low activity against KYC55.
Table 1. Degree of Quorum Sensing Inhibition (QSI) coumaperine derivatives *.
Degree of QSI of
CV026
Degree of QSI of
KYC55
Entry
Monoconjugated-CP
1.
+++
+++
+++
+++
+++
+++
CP-215
CP-270
CP-296
2.
3.
4.
5.
+++
++
CP-282
CP-289
+
+++
6.
7.
++
++
++
++
CP-237
CP-295
8.
+
++
CP-286

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Table 1. Cont.
Degree of QSI of
CV026
Degree of QSI of
Entry
Diconjugated-CP
KYC55
9.
+++
+++
+++
CP-154
CP-158
10.
++
11.
12.
++
+
CP-281 F1
++
++
+
+
CP-38
13.
Entry
14.
CP-9
Degree of QSI of
CV026
Degree of QSI of
KYC55
Triconjugated-CP
++
+
CP-102
* Two tests were carried out using the reporter bacteria: Agrobacterium tumefaciens (KYC55) and Chromobac-
terium 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.
The diconjugated derivative with fluorine substituent CP-154 (Table 1, entry 9) was
highly active in both tests. The acetylated coumaperine CP-158 (Table 1, entry10) was
more effective against CV026 than KYC55. The diacetylated (CP-281-F1) methoxy (CP-38)
and simple phenyl derivatives, CP-9 (Table 1, entries 11–13), displayed moderate and low
activity against CV026 and KYC55, respectively. CP-209 showed low activity against both

Molecules 2021, 26, 2293
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bacterial strains. Coumaperine (CP) CP-32, 147 and 193 exhibited low activity and was
inactive against CV026 and KYC55, respectively. CP-123 and 262-F2 were inactive against
CV026 and displayed low activity against KYC55. The other diconjugated derivatives, such
as CP-10, 27, 50, 184, 194, 209,262-F1 and PIP, were inactive against both the bacteria strains
(Table S1). The triconjugated derivative with the monomethoxy substituent CP-102 (Table 1
entry 14) showed moderate and low QSI against CV026 and KYC55, respectively. The
methylenedioxy-substituted derivative (CP-155) and N,N-dimethyl derivatives (CP-146)
were inactive against both bacterial strains (Table S1).
,
2.4. 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 *.
Compound
Code
Bacillus
subtilis (mm)
Staphylococcus
aureus (mm)
Streptococcus
sobrinus (mm)
Streptococcus
mutans (mm)
Acinetobacter
baumannii (mm)
PA01 (mm)
CP-147
CP-154
CP-9
-
7
8
9
11
9
-
9
9
-
-
-
-
-
-
-
-
-
* Discs with 40 mM of compound in 20
µL solvent (DMSO) were placed on petri dishes with different bacteria. The numbers represent the
diameter of growth inhibition (mm). (-), no activity.
2.5. NF-κB Luciferase Reporter Gene Assay
To further explore the possibility of QS molecules as modulators of NF-
evaluated the effect of the anti-QS active compounds in reducing NF- B activation lu-
ciferase reporter gene NF- B inhibition. The compounds that showed a reduction in NF-
κ
B, we also
κ
κ
κB
activity were further investigated to determine their potency in a dose response experi-
ment and are presented in Figure 5. At these concentrations, the compounds, except for
CP-158, efficiently inhibited NF-
the median inhibitory concentration (IC50), the percent of NF-
against the log of concentrations. CP-154, IC50 = 58.8 M, CP-215, IC50 = 136.37 µM and
CP-286, IC50 = 45.42 M. As a positive control, curcumin was used in two concentra-
tions: 80 and 160 M. Curcumin did not significantly inhibit at 80 M (NF- B activation
is 91.16% ± 6.5%, p = 0.2). At 160 M, curcumin significantly inhibited NF- B (NF-
activation 19.08% ± 10.7%, p = 0.001).
κ
B in L428 cells in a dose-dependent manner. To calculate
κ
B inhibition was plotted
µ
µ
µ
µ
κ
µ
κ
κB
2.6. Quantitation of Activated Nuclear p65-NF-κB Fluorescence
To confirm that the derivatives (CP-154, CP-158, CP-286 and CP-215) inhibit NF-
κ
B,
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).

Molecules 2021, 26, 2293
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Figure 5. Dose response of Nuclear Factor kappa B (NF-
luciferase assay. L428 cells were stably transfected with the NF-
cells were incubated with the compounds at different concentrations for 2 h. Only compounds that
showed NF- B inhibition are shown here. The results represent the percentage of NF-kB activation as
κ
B) activation using the NF-
κ
B reporter gene
κ
B luciferase reporter gene. The
κ
compared to vehicle (DMSO)-treated cells. All samples were normalized to the protein concentration.
Mean + SD, Two-way ANOVA and Tukey’s multiple comparison test. 95% confidence interval
(p-values * ≤ 0.0332, ** ≤ 0.0021 and *** ≤ 0.0002).
Figure 6. NF-
tained with mouse-anti p65 and fluorescent goat anti-mouse IgG. Localization of p65 (
treatment- inactive p65 in the cytoplasm. ( ) After activation with 2.5 ng/mL TNF
activated p65 in the nucleus. All derivatives were added to the cells at 160 M for 120 min in
DMSO (0.16%) then TNF at 2.5 ng/mL was added for 15 min. ( ) Activated cells (strong nuclear
green fluorescence) as a percentage of all the cells in the analyzed field. ( ) Ratio of the mean nu-
κ
B inhibition in A549 cells. A549 cells were fixed in paraformaldehyde and immunos-
) without
for 15 min,
A
B
α
µ
α
C
D
clear/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 *
*** 0.0002). 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 (*).
≤
0.0332, **
≤
0.0021,
≤

Molecules 2021, 26, 2293
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Figure 7. Florescent images of NF-
QS CP derivatives then fixed and immunostained with anti-p65 and anti-mouse IgG conjugate to Alexa AF488 (green)
and nuclear staining with DAPI (blue). ( ) The negative controls were: untreated, treated with the solvent DMSO (0.16%)
for 120 min, TNF (2.5 ng/mL) for 15 min, DMSO (0.16 %) for 120 min with TNF (2.5 ng/mL) for 15 min. ( ) CP-154
and CP-158 at 160 ) CP-286 and CP-215 at 160 M for
120 min with and without TNF ). Curcumin (CU) was used as a positive control with the same
experimental design as in (B) or (C).
κB inhibition. A549 cells were treated with TNFα (2.5 ng/mL) and/or different positive
A
α
α
B
µM for 120 min with and without TNF
α
(2.5 ng/mL) for 15 min. (
C
µ
α
(2.5 ng/mL) for 15 min. (
D
2.7. Cell Viability Assay
We tested the cytotoxicity of the compounds that showed QSI on L428 cells as de-
scribed in Materials and Methods. CP-286, CP-273, CP-296, CP-281-F1, CP-289, CP-270,
CP-282 and CP-215 were not cytotoxic at the concentrations tested. CP-9, CP-154 and
CP-38 were toxic with an LC50 of 24.70
µM, 46.78
µM and 47.44
µM respectively. CP-
158 and CP-295 were less cytotoxic, with LC50 of 285.38
(Supplementary Materials, Table S2)
µM, and 408.27 µM respectively.
3. 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 ap-
plications [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 synthe-
sized compounds were evaluated for their QSI properties. These compounds were then
examined further for their antibacterial activity, cytotoxicity and NF-κB inhibition activity.

Molecules 2021, 26, 2293
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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-3-
oxooctanoyl-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- B in both L428 and A549 cell-lines. In
κ
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 I
Curcumin was used as positive control and significantly inhibit NF-
κ
B, since L428 cells lack IkB, while present in A549 cells.
B activation. The
κ
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.

Molecules 2021, 26, 2293
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Table 3. Summary of Biological activities *.
Compound Code
QSI
Antibacterial
NF-κB Inhibition
Cytotoxic
CP-9
+
+
+
+
+
+
+
−
−
−
+
+
+
+
+
+
+
+
CP-154
CP-158
CP-215
CP-286
−
−
* Five compounds with QSI and NF-
κ
B inhibitory properties were tested for antibacterial and cytotoxic activities.
(+) active (−) inactive.
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-
κ
B
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-
activities (Table 1, Figures 5–8). By contrast, PIP and CP-155 do not inhibit QS or NF-
κ
B inhibition
B. We
κ
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].
Figure 8. Structure activity relationship of methoxy (CP-38) and thiomethyl (CP-147)-diconjugated
coumaperine derivatives.
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 (–SCH3) and in CP-38 a methoxy (–OCH3) 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 communi-
cation. 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.

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4. Materials and Methods
4.1. Chemistry
4.1.1. Materials
All the commercially obtained reagents/solvents for the synthesis of coumaperine
and its derivatives were purchased from Spectrochem® (76/1, Industrial Suburb, Behind
Mysore Sandal Soap Factory, Yeswanthpur, Bangalore-560022, India.), SRL® (608-B, Satellite
Gazebo, Andheri Ghatkopar Link Road, Chakala, Andheri (E), Mumbai-400 099, India.),
Alfa Aesar® (403-404, Delphi ’B’ Wing, Hiranandani Business Park, Powai, Mumbai-400076,
India.), RANKEM® (No. 139, BDA Industrial Suburb, 6th Main, Tumkur Road, Peenya Post,
Bangalore-560058, India.) and Fisher Scientific® (Thermo Fisher Scientific India Pvt. Ltd.,
403-404, Delphi, B Wing, Powai, Hiranandani, Business Park, Maharashtra-400076, India.),
and used as received without further purification. Unless stated otherwise, the reactions
were conducted in oven-dried glassware and under normal atmospheric conditions.
4.1.2. Instrumentation
1H-NMR and 13C-NMR spectra were recorded on Bruker 500 MHz (Billerica, Mas-
sachusetts, USA) spectrometer operating with the 13C resonance frequency of 125 MHz
and the proton resonance frequency of 500 MHz or Bruker 400 MHz spectrometer (Billerica,
Massachusetts, USA) operating with the 13C resonance frequency of 100 MHz and the
proton resonance frequency of 400 MHz. DMSO-d6 or CDCl3 with TMS as an internal
standard was used as an NMR solvent. Data from the 1H-NMR spectroscopy are reported
as chemical shift (δ ppm) with the corresponding integration values. Coupling constants
(J) are reported in Hertz (Hz). Standard abbreviations indicating multiplicity were used as
follows: s (singlet), br (broad), d (doublet), t (triplet), q (quartet) and m (multiplet). Data
from 13C-NMR spectra are reported in terms of chemical shift (
δ ppm). IR spectra were
recorded in Thermo Scientific Nicolet Nexus 470 FT-IR spectrometer (6511 Bunker Lake
Blvd., Ramsey, MN 55303, USA.) and band positions are reported in reciprocal centimeters.
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. Melt-
ing points were recorded with REMI DDMS 2545 (Cama Industrial Estate, Goregaon (East),
Mumbai, India). The instrument is calibrated with benzoic acid before the measurement.
4.1.3. Synthesis Methods
The mono-, di- and triconjugated coumaperine derivatives were synthesized as de-
scribed in detail in the Supplementary Results Section. In short, coumaperine derivatives
with one alkene bond between the aromatic ring and piperidine are termed monoconju-
gated coumaperine derivatives and those with two and three alkene bonds are termed di-
and triconjugated coumaperine derivatives respectively (Figure 9).
Figure 9. Mono-, di- and triconjugated coumaperine derivatives.
The monoconjugated coumaperine derivatives were synthesized in two steps [28,29]
.
Knoevenagel condensation of aldehyde and malonic acid in presence of base yields cin-
namic 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 AlCl3 at room
temperature (Figure 10).

Molecules 2021, 26, 2293
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Figure 10. Synthesis of monoconjugated coumaperine derivatives, CP-237.
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 con-
densed with aromatic aldehydes in presence of base (KOH/t-BuOK) at room temperature
to obtain the final product, di/triconjugated coumaperine derivatives (Figure 3)
4.2. Biological Assays
4.2.1. Quorum Sensing Inhibition Test with Bio-Reporter Chromobacterium violaceum
(CV026) and Agrobacterium tumefaciens (KYC55)
Thin Layer chromatography (TLC) preparation: All derivatives were dissolved in 100%
acetonitrile to a concentration of 40 mM. Then placed 20 µL on the same spot on the TLC
(Merk, Darmstadt, Germany) and let the solvent evaporate before use. CV026: few colonies
were transferred to liquid LB (Difco Lenox medium, Le Pont se Claix, France) for overnight
◦
incubation at 30 C in a 45 degree in orbital shaker (Gerhardt, Königswinter, Germany). At
the O.D. 0.8 3 mL of the starter was added to preheated soft LB agar containing 500 nM
of 3-oxo-C6 (K3007, Sigma-Aldrich, Saint louis, MO, USA). The mixture was then loaded
on the TLC in a glass plate for O.N. incubation in 30 ◦C. The next day, the inhibition zone
was measured, while QS was taking place that produced a bacterial purple pigment, and
when QS is inhibited, no pigment is produced. KYC55 (pJZ372) (pJZ384) (pJZ410): 20
from the frozen stock into 25 mL AT media (AT buffer 20:1.57 M KH2PO4, pH = 7.3. AT
salts mix 20:300 mM (NH4)2SO4, 13 mM MgSO4, 1.4 mM CaCl2, 0.4 mM FeSO47H2O and
0.26 mM MnSO4H2O. 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 g/mL Xgal and 5-nM
µL
×
×
×
×
µ
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
µ
g/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].
4.2.2. 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, Staphy-
lococcus 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.
4.2.3. NF-κB-Luciferase Reporter Gene Assay
L428 cells stable transfectants with the luciferase NF-κB-Luc reporter gene were gen-
erated, as described in Ozer et al. 2009 [35] and were maintained in 500
(G418 disulfate salt, Sigma-Aldrich, Saint louis, MO, USA). The cells (5
µ
g/mL of the G418
×
105/well in tripli-

Molecules 2021, 26, 2293
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cate) were incubated for 2 h in 1 mL of medium containing the solvent (DMSO) or different
concentrations of the molecules tested. Cells were then harvested, lysed and monitored
by a luciferase reporter assay kit (Promega), according to the manufacturer’s instructions.
Measurements were carried out using a luminometer (Promega, GLOMAXTM 20/20 Lu-
minometer, Madison, WI, USA). Data were normalized to the protein concentration in
each lysate as measured by the Bradford method (Bio-Rad) and normalized to the control.
Experiments were repeated at least three times. Initially, we tested all derivatives that
showed QSI activity at two concentrations: 80 and 160
µM (three independent experiments)
for NF- B inhibition activity (data not shown), the dose response of the active derivatives
κ
is shown. Cells maintenance was in RPMI medium supplemented 10% FBS, 1% Glutamine
and Pen-Strep (all from Biological Industries, Bait-Hemed, Israel).
4.2.4. Quantitation of Activated Nuclear p65 Fluorescence
The A549 adherent human lung epithelial cells were grown in DMEM medium with
10% FBS, 1% Glutamine and Pen-Strep (all from Biological Industries, Bait-Hemed, Israel)
and carried by trypsinization (Trypsin EDTA solution B, Biological Industries, Bait-Hemed,
Israel). In contrast to L428 cells, I
κB is present in A549 cells. We quantified the activated
p65 NF- B in the nucleus of the A549 cells as follows: 25,000 cells were plated per well of a
κ
96-well plate (u-clear F-bottom, Greiner Bio-One) and allowed an overnight attachment at
37 ◦C, 5% CO2. The derivatives (CP-154, CP-158, CP-215, CP-286 and Curcumin (CU) as
positive control) were then added at the concentration of 160
sixty micrometers was chosen as the concentration that inhibited NF-
luciferase reporter gene assay. After 2 h, in order to fully activate NF-
Recombinant Human TNF- Protein, R&D system) was added for an additional 15 min.
µ
M for 2 h. One-hundred
B in L428 cells by the
B, TNF (2.5 ng/mL
κ
κ
α
α
Then, the cells were washed twice with 3% FBS in Phosphate Buffered saline (PBS) before
being fixed with 4% paraformaldehyde (16% solution, EM Grade, Electron Microscopy
Sciences) in PBS at room temperature for 20 min. Cells were then washed twice in PBS,
then twice in 3% FBS in PBS before permeabilization with 0.1% Triton
60 min at room temperature. Cells were then washed twice with 3% FBS in PBS before
staining with NF- B subunit P65 (mouse anti-p65 (F-6) SC-8008, Santa Cruz, Dallas, Texas,
USA) diluted 1:50 in 3% FBS and 0.1% Triton 100 in PBS overnight. The cells were then
×
100 in PBS for
κ
×
stained with a secondary antibody AF488 (green) (Goat anti-Mouse IgG (H + L) Alexa
Fluor 488 (A-11029]} Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) diluted
1:400 in 3% FBS and 0.1% Triton
(SouthernBiotech, DAPI Fluoromount-G, blue-nuclear. Birmingham, AL, USA) was then
added for 30 min, followed by 3 washing with PBS. Cell fluorescence was imaged and
quantitated by the Operetta High-Content Imaging System (Perkin Elmer, Waltham, MA,
USA) at 40 magnification. Analysis of the pictures was done through the Columbus
×
100 in PBS overnight at RT. Nuclear staining with DAPI
×
×
server of the company where parameters can be defined, such as cell area, nucleus and
cytoplasm separately. Different cell populations can be distinguished, and scoring was
done on hundreds of cells in each treatment. As an example, see Figure 6. Quantitative
fluorescence intensity was averaged, and a relative fluorescence value was given.
4.2.5. Cell Viability by XTT Assay
The human Hodgkin’s Lymphoma, a derived L428 cell line was used as a model.
The cells were grown in RPMI Medium 1640 with 10% FBS, 1% Glutamine and pen-strep
(all from Biological Industries, Bait-Hemed, Israel). L428 cells do not express the NF-kB
inhibitor of NF-kB (IkB) [37]. As a result, NF-κB is constitutively active and expressed
mainly in the nucleus. 30,000 cells/well were placed in 96-well plates with the coumaper-
ine derivatives at different concentrations or with vehicle (DMSO) to a final volume of
200
µ
L. The plates were incubated for 48 h at 37 ◦C and 5% CO2, followed by the addition
of 50
µ
L of the XTT reagent tetrazolium-formazan, (Biological Industries, Bait-Hemed,
Israel) was added for 2 h at 37 ◦C, 5% CO2. The mitochondria in live cells oxidizes the
tetrazolium-formazan salt generating a color absorbing product at 450 nm. Absorbance

Molecules 2021, 26, 2293
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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 LC50 values of the derivatives, the Probit
transformation was done only on samples with R2 values > 0.85 and only on derivatives
that showed significant cytotoxicity.
4.2.6. 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).
Supplementary Materials: The following supplementary materials are presented: (1) The detailed
synthesis routs and NMR validations of the derivatives. (2) A table of the inactive compounds in the
QSI assay. (3) LC50 cell viability table of active compounds.
Author Contributions: The manuscript was written through contributions of all authors. Concep-
tualization: J.G., A.K. and R.S.K.; methodology; all authors; formal analysis: J.G., A.K. and R.S.K.;
investigation: Y.K., S.M., K.Y. and Y.B.; resources: A.K., J.G. and R.S.K.; writing: Y.K., S.M., J.G., A.K.
and R.S.K.; supervision: A.K., J.G. and R.S.K. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by an Interdisciplinary Faculty Grant BGU. YK was supported
by a fellowship from the Israel Ministry of Science & Technology.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: S.M. thanks VIT Chennai for his Ph.D. fellowship. S.M. and R.S.K. extend
thanks to the Chemistry Division, VIT Chennai, for infrastructure and instrumentation facilities
(CHN analysis, GC–MS and FTIR). The skillful technical assistance from SAIF, VIT Vellore is gratefully
acknowledged. Y.K. thanks her Ph.D. fellowship from the Israel Ministry of Science & Technology.
We thank Esti Kramarsky-Winter for scientific comments and editing. For technical assistance we
also thank Alon Zilha from The Ilse Katz Centre for Meso and Nanoscale Science and Technology
and NIBN, Ben-Gurion University of the Negev, Israel.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
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