Synthesis and anti-inflammatory activities of phloroglucinol-based derivatives

Article abstract of DOI:10.3390/molecules23123232

The natural product phloroglucinol-based derivatives representing monoacyl-, diacyl-, dimeric acyl-, alkylated monoacyl-, and the nitrogen-containing alkylated monoacylphloro- glucinols were synthesized and evaluated for inhibitory activities against the inflammatory mediators such as inducible nitric oxide synthase (iNOS) and nuclear factor kappaB (NF-κB). The diacylphloroglucinol compound 2 and the alkylated acylphloroglucinol compound 4 inhibited iNOS with IC₅₀ values of 19.0 and 19.5 μM, respectively, and NF-κB with IC₅₀ values of 34.0 and 37.5 μM, respectively. These compounds may serve as leads for the synthesis of more potent anti-inflammatory compounds for future drug discovery.

Full text of DOI:10.3390/molecules23123232

molecules
Article
Synthesis and Anti-Inflammatory Activities of
Phloroglucinol-Based Derivatives
Ning Li 1,2, Shabana I. Khan 1,3, Shi Qiu 1 and Xing-Cong Li 1,3,
*
1
Nation Center for Natural Products Research, Research Institute of Pharmaceutical Sciences,
School of Pharmacy, The University of Mississippi, University, MS 38677, USA; ahmulining@163.com (N.L.);
skhan@olemiss.edu (S.I.K.); sqiu@olemiss.edu (S.Q.)
School of Pharmacy, Anhui Medical University, Hefei 230032, China
Department of Biomolecular Sciences, School of Pharmacy, The University of Mississippi, University,
MS 38677, USA
2
3
*
Correspondence: xcli7@olemiss.edu; Tel.: +1-662-915-6742
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 7 November 2018; Accepted: 3 December 2018; Published: 7 December 2018
Abstract: The natural product phloroglucinol-based derivatives representing monoacyl-, diacyl-,
dimeric acyl-, alkylated monoacyl-, and the nitrogen-containing alkylated monoacylphloro- glucinols
were synthesized and evaluated for inhibitory activities against the inflammatory mediators such as
inducible nitric oxide synthase (iNOS) and nuclear factor kappaB (NF-
compound and the alkylated acylphloroglucinol compound inhibited iNOS with IC50 values
of 19.0 and 19.5 M, respectively, and NF- B with IC50 values of 34.0 and 37.5 M, respectively.
κB). The diacylphloroglucinol
2
4
µ
κ
µ
These compounds may serve as leads for the synthesis of more potent anti-inflammatory compounds
for future drug discovery.
Keywords: phlorogluciniol; acylphloroglucinol; anti-inflammatory; iNOS; NF-κB
1
. Introduction
The natural product phloroglucinol (
applications as pharmaceuticals, cosmetics, textiles, paints, and dyes due to their diverse biological
activities [ ]. More than 700 naturally occurring phloroglucinol derivatives have been reported,
of which acylphloroglucinol derivatives comprise the largest group [ ]. Structurally fascinating
acylphloroglucinols possess antidepressant, antimicrobial, antiviral, antitumor, antioxidant, and
anti-inflammatory activities [ ]. The synthetic antispasmodic drug flopropione (3-propionyl-
1) and its derivatives have found a wide range of
1,2
1,3
4–7
phloroglucinol) is a representative of this chemical class [8,9].
The anti-inflammatory activity of the naturally occurring phloroglucinol derivatives acting on
diverse molecular targets has drawn our particular attention. For example, complex phlorotannins
from edible brown algae showed strong inhibitory effects on nitric oxide (NO) production in
lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells [10]. Purified oligomeric phlorotannins
containing fucodiphlorethol A, tetraphlorethol B, tetrafucol A, tetraisofuhalol, and phlorofucofuroeckol
A (Figure 1) also demonstrated the ability to inhibit NO production and suppressing iNOS and
cyclooxygenase (COX)-2 [11]. The acylphloroglucinol hyperforin (Figure 1) from St. John’s wort
suppressed prostaglandin E formation in vitro and in vivo through inhibition of microsomal
2
PGE synthase-1 [12]. 2-Acetyl-4-geranylphloroglucinol (Figure 1) isolated from the Rutaceaous
2
plant Melicope ptelefolia is a strong dual inhibitor of 5-lipoxygenase (LOX) and COX-2 [13].
2
,4-Diacetylphloroglucinol (
antimetastatic activity by mediating inhibition of reactive oxygen species (ROS), nuclear factor kappaB
NF- B), B-cell lymphoma 2 (Bcl-2), matrix metalloproteinase-2 (MMP-2), vascular endothelial growth
2) (Figure 1), a microbial metabolite of Pseudomonas aeruginosa, exhibited
(
κ
Molecules 2018, 23, 3232; doi:10.3390/molecules23123232
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3232
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factor (VEGF) and primary inflammatory mediators such as tumor necrosis factor (TNF)-
α
, interleukin
(IL)-6, IL-1β and NO [14]. In an effort to identify synthetically available phloroglucinol-based
anti-inflammatory compounds, we synthesized several acylphloroglucinols and evaluated their iNOS
and NF-κB inhibitory activities.
Figure 1. Representative phloroglucinol derivatives with anti-inflammatory activities.
2
. Results and Discussion
Based on the concept that both natural and synthetic acylphloroglucinols possess potent biological
activities [
monoacyl-, and the nitrogen-containing alkylated monoacylphloroglucinols were synthesized
Scheme 1). Diacylphloroglucinol ( ) and monoacylphloroglucinol ( ) were prepared first according to
the procedures described previously [18 20]. Using as a starting material, compounds and were
synthesized, respectively, in a one-step reaction adapted from a previously reported procedure [17].
Blocking the two non-hydrogen bonding hydroxyl groups in using MOMCl afforded compound
. This synthesis was intended to assess the role of the hydroxyl groups on the phloroglucinol
6,7,13–17], seven compounds representing monoacyl-, diacyl-, dimeric acyl-, alkylated
(
2
3
–
3
4
6
4
5
ring for activity. The nitrogen-containing compound
7
was designed to alter chemical nature of
23]. It was synthesized by reacting with
-(4-(bromomethyl)benzyl)-piperidine that was prepared from commercially available piperidine
and p-xylylene dibromide in the presence of CH Cl and K CO . As can be seen from Scheme 1, the
the target molecule based on previous reports [21
–
3
1
2
2
2
3
reaction conditions for the synthesis of these compounds were mild as all the reactions were conducted
at room temperature.
The identification of all the synthetic compounds was achieved by interpretation of NMR and
MS data. Compounds
4
–
7
have not been reported prior to this study. It was noted that compound
1
1
7
produced an unusual H-NMR spectrum in CD OD, displaying two sets of H-NMR signals in a
3
ratio of approximately 1.5:1 associated with the para-substituted phenyl ring and some protons of the
piperidine ring (Supplementary Materials). However, when the compound was measured in CDCl
3
1
(
with poor solubility), a single set of H-NMR signals appeared. Use of the weakly basic NMR solvent
1
C D N afforded a H-NMR spectrum showing two sets of signals in a ratio of approximately 4:1 for the
5
5
aromatic protons of the phenyl ring and the methylene attached to the piperidine ring. This interesting
phenomenon can be explained by the fact that compound is, to some extent, a zwitterionic molecule
7

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due to the presence of the acidic phenolic hydroxyl group on the phloroglucinol core and the basic
amine functionality on the side chain. While CD OD can strongly induce the protonation/deuteriation
3
of the nitrogen atom, C D N has the ability to facilitate the formulation of a zwitterion, thereby
5
5
1
13
resulting in the presence of an additional set of NMR signals. The H- and C-NMR signals of
in C D N were unequivocally assigned using 2D NMR of H- H correlation spectroscopy (COSY),
7
1
1
5
5
heteronuclear single quantum coherence (HSQC) and hetero- nuclear multiple bond correlation
HMBC) experiments.
(
Scheme 1. Synthesis of compounds 2–7. Reagents and conditions: (i) nitrobenzene, AlCl , rt for 0.5 h,
3
◦
then isobutyryl chloride, 65 C for 21 h; (ii) NaOH, H O, TBAI, 1-bromo-4-(bromomethyl)benzene,
2
hexane, rt for 48 h; (iii) acetone, K CO , CH OCH Cl, rt for 24 h; (iv) NaOH, H O, TBAI, p-xylylene
2
3
3
2
2
dibromide, toluene, rt for 6 h; (v) piperidine, CH Cl , K CO , p-xylylene dibromide, NaOH, H O,
2
2
2
3
2
MeOH, rt for 7 h.
In vitro anti-inflammatory testing of compounds
2
–7
was conducted by assessing the inhibition
of iNOS in LPS-induced macrophages (RAW 264.7) and inhibition of NF-
κ
B in phorbol 12-myristate
13-acetate (PMA)-induced human chondrosarcoma cells (SW1353). As shown in Table 1,
diacylphloroglucinol compound
2 showed the best activity, inhibiting iNOS and NF-κB with IC50
values of 19.0 and 34.0
values of 2.5 and 20.5 M, respectively. The activity of alkylated acylphloroglucinol
targets (IC50 19.5 and 37.5 M, respectively) was also very similar to compound . When the hydroxyl
groups in were protected leading to compound , iNOS inhibitory activity was decreased (IC ,
M) while NF- B activity was slightly increased (IC , 29.0
groups play a role for the activity and the differential activity between the two compounds may be
associated with molecular lipophilicity. Dimeric acylphloroglucinol inhibited iNOS to a similar extent
M). The nitrogen- containing compound
was only moderately inhibiting iNOS with an IC50 value of 49.0 M, indicating the introduction of
was
M, indicating that additional acylation or
alkylation on the phloroglucinol ring is necessary to enhance anti-inflammatory activity. Cytotoxicity
µM, respectively, compared with the positive control parthenolide with IC50
µ
4
against these two
µ
2
4
5
5
0
5
0
µ
κ
µM). This suggests these two hydroxyl
50
6
(
IC , 19.0 µM) but inhibition of NF-κB was weaker (IC , 85.0 µ
50 50
7
µ
the amino group in this chemotype is not helpful. On the other hand, monoacylphloroglucinol
only marginally inhibiting NF- B with an IC50 value of 90.0
3
κ
µ
testing using African monkey kidney fibroblast cell line (Vero) showed that compounds
2
–7
were not
cytotoxic up to a concentration of 50 µM
The anti-inflammatory activity observed for compound
to be consistent with the previously reported data for the analogue 2,4-diacetylphloroglucinol [13].
Compound was previously reported to possess potent in vitro antifungal activity against Cryptococcus
2 against iNOS and NF-κB appears
2
neoformans [17]. An enhanced inflammatory cell response in C. neoformans infection has been observed
via regulation of IL-23 and IL-12 [24]. Our findings suggest that the anti-inflammatory activity of
compound 2 targeting iNOS and NF-κB would have a positive impact on its antifungal activity.

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Table 1. Anti-inflammatory activity of synthetic acylphloroglucinols.
iNOS Inhibition
IC , µM)
NF-κB Inhibition
(IC50, µM)
Compound
(
5
0
2
3
4
5
6
7
19.0 ± 1.4
34.0 ± 8.5
90.0 ± 14.1
37.5 ± 3.5
29.0 ± 15.5
85.0 ± 21.2
NA
NA
19.5 ± 0.7
50.0 ± 0
19.0 ± 1.4
49.0 ± 1.4
2.5 ± 0.2
Parthenolide
20.5 ± 2.9
NA, not active at 50 µM for iNOS and 100 µM for NF-κB.
In conclusion, the natural product phloroglucinol-derived compounds were synthesized and
evaluated for anti-inflammatory activity against iNOS and NF- B as well as cytotoxicity against the
κ
mammalian Vero cells. This exploratory study has generated meaningful structure activity relationship
information from the limited number of synthetic compounds. The diacylphloroglucinol compound
2
and the alkylated monoacylphloroglucinol compound 4 are dual inhibitors of iNOS and NF-κB.
Further synthesis of these two chemotypes of compounds may afford compounds with improved
anti-inflammatory activities which could be better candidates for future drug discovery.
3
. Materials and Methods
3
.1. General Experimental Procedures
The nuclear magnetic resonance (NMR) spectra using standard pulse programs were recorded at
room temperature on a BrukerAvance DPX-400 spectrometer (Bruker, Billerica, MA, USA) operating at
1
13
4
00 ( H) and 100 ( C) MHz. The chemical shift (δ, ppm) values were calibrated using the residual
NMR solvent and coupling constant (J) was reported in Herts (Hz). High-resolution time-of-flight
mass spectrometry (TOF-MS) and electrospray ionisation mass spectrometry (ESI-MS) were measured
on an Agilent series 1100 SL spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with
an ESI source. Thin layer chromatography (TLC) was performed on silica gel aluminum sheets (silica
gel 60 F254, Merck, Darmstadt, Germany) and visualized by UV 254 nm and spraying 10% H SO
2
4
followed by heating. Column chromatography was done on normal-phase silica gel (230 × 400 mesh
,
J. T. Baker, Center Valley, PA, USA) and Sephadex LH-20 (75 m, GE Healthcare Bio-Sciences
µ
AB, Uppsala, Sweden). Reactants and reagents including anhydrous phloroglucinol, nitrobenzene,
aluminum chloride (AlCl ), n-butyl ammonium iodide (TBAI), 1-bromo-4-bromomethyl-benzene,
3
chloromethoxymethane (CH OCH Cl), toluene, and piperidine were purchased from Sigma-Aldrich
3
2
(
St. Louis, MO, USA) in appropriate grades and were used without further purification. The yield of
1
13
each synthetic product after column chromatography is reported. The H- and C-NMR assignments
for all synthetic products and key intermediates with appropriate numbering systems are shown in the
Supplementary Materials. Compounds
previously [18 20] and their identification was made by comparison of their NMR spectroscopic data
see Supplementary Materials) with those reported in the literature [17].
2 and 3 were synthesized according to the protocols described
–
(
3
.2. Synthesis of Compound 4
To a solution of compound 3 (3.0 g), NaOH (1.01 g), and TBAI (catalyzer, 500 mg) in H O (225 mL)
2
was added 1-bromo-4-(bromomethyl)benzene (2.5 g) in hexane (20 mL) dropwise. The mixture was
stirred at room temperature for 48 h. The precipitated crude product was collected by filtration.
The residue was purified by chromatography on silica gel using acetone:hexane (1:2) to afford
yield 30.0%).
4 (1.67 g,

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1
1
-[3-(4-Bromo-benzyl)-2,4,6-trihydroxy-phenyl]-2-methyl-propan-1-one (
4
).
H-NMR (400 MHz,
0
0
CD COCD ) δ: 14.33 (s, OH), 7.35 (2H, d, J = 8.2 Hz, H-14,14 ), 7.24 (2H, d, J = 8.2 Hz, H-13, 13 ), 6.16
1H, s, H-4), 3.99 (1H, m, H-8), 3.85 (2H, s, H-11), 1.13 (6H, d, J = 6.6 Hz, H-9, 10). C-NMR (100 MHz,
: 211.0 (C-7), 165.7 (C-1), 162.8 (C-5), 160.7 (C-3), 142.4 (C-12), 131.6 (C-14, 14 ), 131.5
C-13, 13 ), 119.4 (C-15), 107.3 (C-6), 104.2 (C-2), 95.2 (C-4), 39.5 (C-8), 28.0 (C-11), 19.6 (C-9, 10). MS
3
3
13
(
0
CD COCD )
δ
3
3
0
(
ESI-(−) m/z = 363.0/365.2 [M − H]− for C H BrO .
17
17
4
3
.3. Synthesis of Compound 5
A suspension of compound
4 (346 mg) and K CO (2.76 g) in acetone (35 mL) was stirred for
2 3
3
0 min, then CH OCH Cl (480 mg) was added. After stirring at room temperature for 48 h, the reaction
3 2
mixture was filtered. The filtrate was concentrated, and the residue was purified by chromatography
on silica gel with acetone:hexane (1:12) to give 5 (239 mg, yield 52.8%).
1
1
-[3-(4-Bromo-benzyl)-2-hydroxy-4,6-bis-methoxymethoxy-phenyl]-2-methyl-propan-1-one (
5
). H-NMR (400
0
0
MHz, CDCl3)
δ
: 14.0 (s, OH), 7.35 (2H, d, J = 8.4 Hz, H-14, 14 ), 7.21 (2H, d, J = 8.4 Hz, H-13, 13 ), 6.46
(
1H, s, H-4), 5.28 (2H, s, -OCH ), 5.24 (1H, s, -OCH ), 3.93 (2H, s, H-11), 3.82 (1H, m, H-8), 3.55 (3H, s,
2 2
13
-OCH ), 3.41 (3H, s, -OCH ), 1.21 (6H, d, J = 6.7 Hz, H-9, 10). C-NMR (100 MHz, CDCl )
δ
: 210.7
3
3
3
0
0
(
C-7), 164.0 (C-5), 160.5 (C-1), 158.9 (C-3), 140.6 (C-12), 131.0 (C-14, 14 ), 130.4 (C-13, 13 ), 119.2 (C-15),
1
2
10.7 (C-2), 106.0 (C-4), 94.9 (-OCH ), 94.0 (-OCH ), 91.4 (C-6), 56.8 (-OCH ), 56.5 (-OCH ), 39.8 (C-8),
2 2 3 3
−
7.7 (C-11), 19.4 (C-9, 10). MS ESI-(−) m/z = 451.0/453.2 [M − H] for C H BrO .
21
25
6
3
.4. Synthesis of Compound 6
A suspension of compound
3 (98 mg) and NaOH (22 mg) in H O (7.5 mL) was stirred about
2
30–40 min until it became clear. To this solution was added TBAI (9.8 mg) and p-xylylene dibromide
(144 mg) in toluene (4.5 mL) dropwise. After stirring at room temperature for 6 h, the reaction mixture
was extracted with ethyl acetate (EtOAc) (3 20 mL). The combined EtOAc layer was concentrated
×
under reduced pressure to afford a residue, which was purified by chromatography on silica gel using
acetone:hexane (1:2) to give 6 (44 mg, yield 17.8%).
2
-Methyl-1-{2,4,6-trihydroxy-3-[4-(2,4,6-trihydroxy-3-isobutyryl-benzyl)-benzyl]-phenyl}-propan-1-one (
6
).
1
0
0
0
H-NMR (400 MHz, CD COCD )
4
MHz, CD COCD )
δ
: 14.19 (s, OH), 7.17 (4H, s, H-13, 13 , 14, 14 ), 6.10 (2H, s, H-4, 4 ),
3
3
0
0
0
0
13
.02 (2H, m, H-8, 8 ), 3.85 (4H, s, H-11, 11 ), 1.14 (12H, d, J = 6.8 Hz, H-9, 9 , 10, 10 ). C-NMR (100
0
0
0
0
0
δ
: 210.1 (C-7, 7 ), 164.8 (C-1, 1 ), 162.0 (C-5, 5 ), 159.7 (C-3, 3 ), 138.9 (C-12, 12 ),
3
3
0 0 0 0 0 0 0
128.1 (C-13, 13 , 14, 14 ), 107.3 (C-6, 6 ), 103.4 (C-2, 2 ), 94.3 (C-4, 4 ), 38.7 (C-8, 8 ), 28.5 (C-11, 11 ), 18.8
0
0
−
(C-9, 9 , 10, 10 ). MS ESI-(−) m/z = 493.2 [M − H] for C H O .
28
30
8
3
.5. Synthesis of Compound 7
To a solution of piperidine (2.55 g) in CH Cl (100 mL) was added K CO (5.46 g). After stirring
2
2
2
3
at room temperature for 1 h, p-xylenedibromide (7.86 g) in CH Cl (100 mL) was added dropwise, and
2
2
the reaction continued for 3 h. The reaction mixture was filtered. The filtrate was concentrated under
reduced pressure, and the residue was purified by chromatography on silica gel using acetone:hexane
(
1:7) to yield the intermediate 1-(4-(bromomethyl) benzyl)piperidine (2.0 g).
To a solution of compound (196 mg) and NaOH (44 mg) in H O (60 mL) was added the
intermediate (707 mg) in MeOH (40 mL) dropwise. After stirring at room temperature for 4 h, the
reaction mixture was extracted with EtOAc (3 80 mL) and concentrated under reduced pressure.
The residue was purified by chromatography on silica gel using CHCl :MeOH:acetone (10:1:1) to yield
3
2
×
3
7
(35 mg, yield 9.2%).
2
-Methyl-1-[2,4,6-trihydroxy-3-(4-piperidin-1-ylmethyl-benzyl)-phenyl]-propan-1-one (7
). 1H-NMR (400
0
0
MHz, C D N)
4
δ
: 7.74 (2H, d, J = 6.0 Hz, H-13, 13 ), 7.68 (2H, d, J = 6.0 Hz, H-14, 14 ), 6.61 (1H, s, H-4),
5
5
0
0
.37 (2H, s, H-11), 4.35 (1H, m, H-8), 4.23 (2H, s, H-16), 2.97 (4H, m, H-17, 17 ), 1.77 (4H, m, H-18, 18 ),

Molecules 2018, 23, 3232
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1
.29 (6H, d, J = 6.7 Hz, H-9, 10), 1.28 (2H, m, H-19). 13C-NMR (100 MHz, C D N)
δ
: 211.2 (C-7), 166.4
5
5
0 0
(
C-1), 164.6 (C-3), 162.5 (C-5), 145.2 (C-12), 132.16 (C-14, 14 ), 130.3 (C-13, 13 ), 127.89 (C-15), 107.5 (C-6),
0
0
1
04.8 (C-2), 95.9 (C-4), 61.0 (C-16), 52.99 (C-17, 17 ), 39.7 (C-8), 29.2 (C-11), 23.62 (C-18, 18 ), 22.78 (C-19),
+
2
0.3 (C-9, 10). HRTOF-MS ESI-(+) m/z = 384.2170 [M + H] for C H NO (cal. 384.2176).
23
30
4
1
0
7 (400 MHz, C D N) δ: 7.78 (2H, d, J = 5.8 Hz, H-13, 13 ), 7.63 (2H, d, J
5 5
H-NMR for the isomer of
0
=
5.8 Hz, H-14, 14 ), 6.61 (1H, s, H-4), 4.97 (2H, s, H-16), 4.37 (2H, s, H-11), 4.35 (1H, m, H-8), 2.97 (4H,
0
0
13
m, H-17, 17 ), 1.77 (4H, m, H-18, 18 ), 1.36 (2H, m, H-19), 1.29 (6H, d, J = 6.7 Hz, H-9, 10). C-NMR
for the isomer of
7
(100 MHz, C D N)
δ
: 211.2 (C-7), 166.4 (C-1), 164.6 (C-3), 162.5 (C-5), 145.2 (C-12),
5
5
0
0
1
32.23 (C-14, 14 ), 130.3 (C-13, 13 ), 128.04 (C-15), 107.5 (C-6), 104.8 (C-2), 95.9 (C-4), 64.3 (C-16), 53.25
0
0
(C-17, 17 ), 39.7 (C-8), 29.2 (C-11), 23.84 (C-18, 18 ), 23.01 (C-19), 20.3 (C-9, 10).
3
.6. Assay for iNOS Inhibition
The assay was performed in mouse macrophages (RAW264.7, obtained from ATCC)) cultured in
phenol red free RPMI medium supplemented with 10% bovine calf serum and 100 U/mL penicillin
◦
G sodium, and 100
µ
g/mL streptomycin at 37 C in an atmosphere of 5% CO and 95% humidity.
2
Cells were seeded in 96-well plates (50,000 cells/well) and incubated for 24 h for a confluency of 75%
or more. Test samples diluted in serum free medium were added, and after 30 min of incubation, LPS
(5
µg/mL) was added and cells were further incubated for 24 h. The concentration of nitric oxide
(NO) was determined by measuring the level of nitrite in the cell culture supernatant by using Griess
reagent. Percent inhibition of nitrite production by the test compound was calculated in comparison to
the vehicle control. IC50 values were obtained from concentration response curves. Parthenolide was
used as a positive control [25].
3
.7. Assay for NF-κB Inhibition
The assay was performed in human chondrosarcoma cells (SW1353, obtained from American Type
Culture Collection (ATCC), Manassas, VA, USA). Cells were cultured in 1:1 mixture of DMEM/F12
supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin G sodium, and 100
µ
g/mL
10 ) were washed
µL of antibiotic-free
◦
7
streptomycin at 37 C in an atmosphere of 5% CO and 95% humidity. Cells (1.2
×
2
once in an antibiotic and FBS-free DMEM/F12, and then resuspended in 500
DMEM/F12 containing 2.5% FBS. NF-κB luciferase plasmid construct was added to the cell suspension
at a concentration of 50 g/mL and incubated for 5 min at room temperature. The cells were
µ
electroporated at 160 V and one 70-ms pulse using BTX disposable cuvettes model 640 (4-mm gap) in
a BTX Electro Square Porator T 820 (BTX I, San Diego, CA, USA). The transfected cells were plated
5
to the wells of 96-well plates at a density of 1.25
×
10 cells per well. After 24 h, cells were treated
with different concentrations of test compound for 30 min before the addition of PMA (70 ng/mL)
and incubated for 8 h. Luciferase activity was measured using the Luciferase Assay kit (Promega).
Light output was detected on a SpectraMax plate reader. Percent inhibition of luciferase activity was
calculated as compared to vehicle control, and IC50 values were obtained from concentration response
curves. Parthenolide was used as positive control [25].
3
.8. Cytotoxicity Assay
Cytotoxicity was determined against the mammalian cell line Vero (African green monkey
kidney fibroblast) which was obtained from ATCC. The detailed assay procedure has been described
previously [26]. In brief, cells were seeded in 96-well plates (10,000 cells /well), and after 24 h of
incubation, they were treated with various dilutions of test samples for 48 h. The cell viability was
determined by tetrazolium dye WST-8. Doxorubicin was included as drug control.
Supplementary Materials: The following are available online. Supplementary Materials, including MS, HRMS,
1
D and 2D NMR spectra for the synthetic compounds, are available online.

Molecules 2018, 23, 3232
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Author Contributions: N.L.: chemical synthesis, data analysis and manuscript preparation; S.I.K.: bioassay
and interpretation of data and manuscript editing; S.Q.: NMR analysis; X.-C.L.: project design and
manuscript preparation.
Funding: This work was supported by the USDA Agricultural Research Service Specific Cooperative Agreement
No. 58-6060-6-015.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available.
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