Antioxidative and anti-inflammatory activity of psiguadial B and its halogenated analogues as potential neuroprotective agents

Article abstract of DOI:10.1016/j.bioorg.2021.105027

Psiguadial B (8), and its fluoro- (8a), chloro- (8b), and bromo- (8c) derivatives were synthesized using a sodium acetate-catalyzed single step coupling of three components: β-caryophyllene (5), diformylphloroglucinol (11), and benzaldehyde (12). These co

Full text of DOI:10.1016/j.bioorg.2021.105027

Bioorganic Chemistry 113 (2021) 105027
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Antioxidative and anti-inflammatory activity of psiguadial B and its
halogenated analogues as potential neuroprotective agents
,
,
,
,
Tara Man Kadayata 1, Dong Eun Kimb 1, Sang Bong Leec 1, Kyungjin Junga 1, Sang Eun Parkb,
Ji-Ye Hongb, Jina Kima, Aarajana Shresthaa, Dong-Su Kimd, Hongchan Ana, Nayeon Kima,
Su-Jeong Leea, Sugyeong Kwona, Suhui Kima, Jun Yeon Hwanga, Shinae Kima,
Dongyup Hahne, Hyukjae Choif, Sang-Jip Namg, Yong Hyun Jeonh, Jung Jin Hwangb
,
,i,*
Sung Jin Choj , Jungwook China
,
*
,*
a New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea
b Asan Institute for Life Sciences, Asan Medical Center, Seoul 05505, Republic of Korea
c Vaccine Commercialization Center, Gyeongbuk Institute for Bio Industry, Andong 33618, Republic of Korea
d Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology, 34114 Daejeon, Republic of Korea
e School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea
f College of Pharmacy, Yeungnam University, Gyeongbuk 38541, Republic of Korea
g Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760, Republic of Korea
h Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea
i Department of Convergence Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea
j Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Psiguadial B (8), and its fluoro- (8a), chloro- (8b), and bromo- (8c) derivatives were synthesized using a sodium
acetate-catalyzed single step coupling of three components: β-caryophyllene (5), diformylphloroglucinol (11),
and benzaldehyde (12). These compounds efficiently and dose-dependently decreased H2O2-induced cell death, a
quantitative marker of cell death, in primary cultures of mouse cortical neurons. Psiguadial B also decreased
neuronal death and accumulation of ROS induced by FeCl2 in cortical cultures. The in vitro effects of these
Psiguadial B
Reactive oxygen species
Antioxidative effect
Anti-inflammatory effect
Neuroprotective agent
Short synthetic route
compounds in lipopolysaccharide (LPS)-induced expression of nitric oxide (NO), and TNF-α and IL-6 by sup-
pressing the NF-κB pathway in immune cells demonstrated their antioxidative and anti-inflammatory activity.
The present findings warrant further research on the development of psiguadial B-based neuroprotective agents
for the treatment of neurodegenerative diseases, acute brain injuries and immunological disorders.
1. Introduction
meroterpenoids (Fig. 1C), psiguadial A (7), psiguadial B (8), psidial A
(9), and guajadial (10) against doxorubicin-resistant and -sensitive
Psidium guajava L., commonly known as guava, is an edible fruit tree
cultivated in subtropical and tropical regions has traditionally been used
in several conditions such as diarrhea, dysentery, hypertension and
hyperglycemia [1,2]. Several researchers have reported that guava
contains pharmacologically active constituents including flavonoids
[3–5], triterpenoids [6,7], and sesquiterpenoids (Fig. 1A and 1B)
[8–10]. A group of Chinese researchers have isolated several new
sesquiterpenoid-based meroterpenoids from guava leaves [9,11–15].
Shao et al. first reported isolation and antiproliferative activities of four
human hepatoma cells, HepG2/ADM and HepG2, respectively [9].
Among them, psiguadial B (8) showed the most potent inhibitory ac-
tivity (IC50 = 45 ± 1.41 nM) against HepG2 cells. Despite the potent
cytotoxic effect of psiguadial B in preliminary testing, further biological
studies of this compound had not been reported due to synthetic chal-
lenges of the sample. From a synthetic chemistry point of view, it was
challenging to synthesize psiguadial B (8), which contains a bicyclo
[4.3.1]decane ring, transfused to a highly functionalized chromane and
cyclobutane. In 2016, Chapman et al. reported the first 16 steps of the
* Corresponding authors.
E-mail addresses: jjhwang@amc.seoul.kr (J. Jin Hwang), sjcho@kist.re.kr (S. Jin Cho), jwchin@dgmif.re.kr (J. Chin).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.105027
Received 27 October 2020; Received in revised form 29 April 2021; Accepted 24 May 2021
Available online 26 May 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

T. Man Kadayat et al.
Bioorganic Chemistry 113 (2021) 105027
Fig. 1. Structures of active constituents isolated from leaves of Psidium guajava.
Scheme 1. Preparation of psiguadial B (8) and its halogenated analogues 8a-c.
enantioselective total synthesis of psiguadial B [16]. However, this long
synthetic method resulted in overall yield less than 1% of compound 8,
making it quantitatively insufficient for further biological testing.
Therefore, developing a new, concise and efficient synthetic method to
synthesize psiguadial B was needed. As part of our major program to find
a new biological activity of psiguadial B, in May of 2017, we first
attempted to synthesize psiguadial B via short biomimetic synthetic
route (see Official E-Notebook proof of our work in Supplementary Data)
as proposed in Scheme 1 based on report of guajadial and psidial A
synthesis by Lawrence et al [17]. Concurrently, Cramer and co-workers
[18] previously reported a single step N,N’-dimethylethylenediamine
(DMEDA)–catalyzed synthesis of psiguadial B, while they reported zero
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T. Man Kadayat et al.
Bioorganic Chemistry 113 (2021) 105027
yield in the presence of NaOAc with different reaction solvent and
conditions than our work.
2.2. Synthetic procedures and compound characterization data
Previous studies have shown that constituents of guava have various
therapeutic activities including hepato-protective [19], anti-
hyperglycemic [20,21], anti-inflammatory [22], antimicrobial [23] and
antioxidant [24]. There are limited studies on effect of guava constitu-
ents against oxidative stress and neurodegeneration [25–27]. Neuro-
degenerative diseases such as Parkinson’s disease and Alzheimer’s
disease are characterized by progressive loss of function and structure of
neurons in the brain or spinal cord [28]. The etiology of neurodegen-
erative diseases are environmental and genetic factors, however, the
oxidative stress due to generation of reactive oxygen species such as
pathogenic superoxide anion (O.2ꢀ ) and hydroxyl (HO.) free radical are
considered to be a major factor of the neurodegenerative process
[29,30]. It has been reported that high level of redox-active metal ions
such as Fe2+ and Cu2+ promote neurodegeneration by generating HO.
free radicals from hydrogen peroxide (H2O2) [31–33]. Recently, studies
have shown the interrelation between oxidative stress and inflammation
in neurodegeneration [34,35]. Furthermore, several studies have re-
ported that the presence of halogen functionalities in compound is
important for improved therapeutic effect by inhibiting reactive oxygen
species (ROS) production, which might be due to enhanced physio-
chemical and pharmacokinetic properties of compounds [36,37].
Based on these studies, we anticipate that psiguadial B and its haloge-
nated analogues would exhibit neuroprotective and anti-inflammatory
activity through their antioxidative effects. Here we report, for the
first time, sodium acetate (NaOAc)-catalyzed short synthesis psiguadial
B and its halogenated analogues 8a-c as novel and potential neuro-
protective agents for the treatment of neurodegenerative diseases and
acute brain injuries.
2.2.1. Preparation of diformylphloroglucinol (11)
To a stirred solution of N,N-dimethylformamide (14, 1.23 mL, 15.86
mmol, 2.0 equiv.), phosphorus oxychloride (15, 1.62 mL, 17.45 mmol,
2.2 equiv.) was added dropwise at room temperature under a nitrogen
atmosphere and the mixture was continued for 30 min to obtain Vils-
meier reagent (16). Then to a stirred solution of anhydrous phlor-
oglucinol (1.0 g, 7.93 mmol, 1.0 equiv.) in dioxane (5 mL) reagent 16
(~2 equiv.) was slowly added via cannula. This solution was then stirred
for 12 h at room temperature, under a nitrogen atmosphere. After 12 h a
yellow amorphous solid was formed. This solid mixture was cooled to
◦
0 C and then added to an ice-water slurry (30 mL). The mixture was
stirred for 4 h at room temperature which resulted formation of cream
precipitate. Upon filtering and washing with more water, solid was
suspended in 8 mL water and refluxed for 5 min. After cooling to 0 ◦C, a
salmon colored solid was formed which was filtered off and washed with
cold water (15 mL). The solid was dried under vacuum for 20 h at 90 ◦C
to get diformylphloroglucinol (11, 1.062 g, 73.5%) as a salmon pink
colored solid. ESI LC/MS: m/z calcd. for C8H6O5 [M + H]+: 183.03;
found 183.25. 1H NMR (400 MHz, DMSO‑d6) δ 13.51 (br, 1H, OH),
12.49 (br s, 2H, OH), 10.01 (s, 2H, CHO), 5.90 (s, 1H). 13C NMR (100
MHz, DMSO‑d6) δ 191.86 (2C), 169.88 (2C), 169.49, 104.23 (2C), 94.54.
2.2.2. Preparation of (+)-Psiguadial B (8)
To a stirred solution of diformylphloroglucinol (11, 100 mg, 0.549
mmol) and sodium acetate (4.5 mg, 0.06 mmol, 0.1 equiv.) in acetic acid
(4 mL), benzaldehyde (12, 0.11 mL, 1.10 mmol, 2.0 equiv.) and β-Car-
yophyllene (5, 0.37 mL, 1.65 mmol, 3.0 equiv.) were added dropwise at
80 ◦C, the mixture was continuingly stirred for 24 h at 80 ◦C. Upon
cooling to room temperature, brine (5 mL) and diethyl ether (Et2O, 20
mL) were added and the layers separated. The aqueous layer was then
extracted with Et2O (10 mL × 3), and the combined organic layers were
washed with brine (20 mL) and dried over anhydrous Na2SO4. It was
filtered and concentrated to give the crude residue, which was purified
by preparative HPLC (40–72% ACN in H2O) to give product (8) as a pale
cream solid (14 mg, 5.37%). ESI LC/MS: m/z calcd. for C30H34O5 [M +
H]+: 475.24; found 475.09. 1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H,
OH), 13.02 (s, 1H, OH), 10.06 (s, 2H, CHO), 7.26–7.18 (m, 3H),
7.17–7.09 (br m, 2H), 3.47 (d, J = 11.48 Hz, 1H), 2.15 – 2.06 (m, 2H),
1.95–1.87 (m, 1H), 1.84–1.77 (m, 1H), 1.73–1.65 (m, 3H), 1.50–1.44
(m, 3H), 1.42–1.29 (m, 3H), 1.26–1.23 (m, 1H), 1.06–1.02 (m, 1H), 0.99
(apparent d, 6H), 0.84 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 192.36,
191.53, 169.66, 168.53, 163.51, 143.42, 128.18 (3C), 126.23 (2C),
105.73, 104.65, 104.15, 84.14, 50.02, 47.46, 44.04, 40.39, 37.61,
36.93, 35.44, 35.09, 33.46, 30.63, 29.37, 26.09, 23.91, 20.75, 20.11.
2. Materials and methods
2.1. Chemistry
All commercially available reagent-grade chemicals were used as
received without further purification. Reactions were monitored by LC/
MS, and thin-layer chromatography (TLC), performed using 0.2 mm
silica gel plates (Merck 60 F254) and visualized by UV light (254 nm).
Column chromatography was performed using a CombiFlash Rf system
with RediSep Rf (Teledyne Isco, Lincoln, NE). Target compounds were
purified by preparative HPLC on Kinetex 5 μm biphenyl, 100 Å column
(GX-281 HPLC system, Gilson, Middleton, WI, USA; column tube 250
mm × 21.2 mm i.d.), with acetonitrile and water as binary gradient
elution. Mobile phase A was water with 0.1% trifluoroacetic acid, and
mobile phase B was acetonitrile with 0.1% trifluoroacetic acid. The in-
jection volum was 1 mL and a gradient of 40–70% B was run at a flow
rate of 17 mL/min over 60 min. The purity of target compounds was
determined to be above 95% by analytical HPLC using dual different
wavelength UV detector (254 nm and 280 nm). Bruker Nuclear magnetic
resonance (NMR) spectrometer was used to record for 1H and 13C
spectra at 400 MHz and 100 MHz, respectively. Compounds were dis-
2.2.3. Preparation of fluoro (+)-Psiguadial B (8a)
To a stirred solution of diformylphloroglucinol (11, 100 mg, 0.549
mmol) and sodium acetate (4.5 mg, 0.06 mmol, 0.1 equiv.) in acetic acid
(4 mL), 4-fluorobenzaldehyde (12a, 0.12 mL, 1.10 mmol, 2.0 equiv.)
and β -Caryophyllene (5, 0.37 mL, 1.65 mmol, 3.0 equiv.) were added
dropwise at 80 ◦C, the mixture was continuingly stirred for 24 h at 80 ◦C.
Upon cooling to room temperature, brine (5 mL) and diethyl ether (20
mL) were added and the layers separated. The aqueous layer was then
extracted with diethyl ether (10 mL × 3), and the combined organic
layers were washed with brine (20 mL) and dried over anhydrous
Na2SO4. It was filtered and concentrated to give the crude residue,
which was purified by Preparative HPLC (40–72% ACN in H2O) to
giveproduct (8a) as a white solid (18 mg, 6.66%). ESI LC/MS: m/z calcd.
◦
solved in DMSO‑d6 or CDCl3, and the spectra were recorded at 25 C.
Chemical shifts (δ) were reported in parts per million (ppm) relative to
tetramethylsilane (TMS) as an internal standard and coupling constants
were expressed in hertz (Hz). Mass spectra were recorded with a positive
electrospray ionization (ESI) mode on LCMS-2020 system (Shimadzu,
Tokyo, Japan) and Thermo Scientific Dionex Ultimate 3000 system.
1
for C30H33FO5 [M + H]+: 493.24; found 493.65. H NMR (400 MHz,
3

T. Man Kadayat et al.
Bioorganic Chemistry 113 (2021) 105027
CDCl3) δ 13.50 (s, 1H, OH), 13.05 (s, 1H, OH), 10.07 (s, 2H, CHO), 7.04
(br s, 2H), 6.96–6.92 (m, 2H), 3.47 (d, J = 11.48 Hz, 1H), 2.15–2.07 (m,
2H), 1.95–1.78 (m, 2H), 1.70–1.57 (m, 3H), 1.51–1.38 (m, 5H),
1.36–1.31 (m, 1H), 1.27–1.24 (m, 1H), 1.08–1.03 (m, 1H), 1.01
(apparent d, 6H), 0.86 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 192.32,
191.58, 169.58, 168.55, 163.41, 162.56, 160.13, 139.02, 138.99,
128.91, 115.13, 114.92, 105.53, 104.63, 104.17, 84.15, 50.09, 47.44,
44.05, 39.69, 37.59, 36.95, 35.44, 35.08, 33.48, 30.61, 29.37, 26.06,
23.87, 20.73, 20.12.
ICR mice on feeder astrocytic cultures and maintained in Dulbecco’s
modified Eagle’s medium (Gibco) supplemented with 5% fetal bovine
serum (Hyclone), 5% horse serum (Gibco), 2 mM glutamine (Sigma),
and 1% penicillin/streptomycin (Cambrex).
2.3.2. Lactate degydrogenase (LDH) assay
Lactate dehydrogenase (LDH) released into culture media was
assayed in potassium phosphate buffer containing 23 mM pyruvate and
0.3 mg/ml β-NADH by monitoring the conversion of NADH to NAD + at
340 nm in a spectrophotometer (Molecular Devices, Spectramax). LDH
value was scaled to the mean value of sister cultures after 24 h of
2.2.4. Preparation of chloro (+)-Psiguadial B (8b)
To a stirred solution of diformylphloroglucinol (11, 100 mg, 0.55
mmol) and sodium acetate (4.5 mg, 0.06 mmol, 0.1 equiv.) in acetic acid
(4 mL), 4-chlorobenzaldehyde (12b, 154 mg, 1.10 mmol, 2.0 equiv.) and
β -Caryophyllene (5, 0.37 mL, 1.65 mmol, 3.0 equiv.) were added
dropwise at 80 ◦C, the mixture was continuingly stirred for 24 h at 80 ◦C.
Upon cooling to room temperature, brine (5 mL) and diethyl ether (20
mL) were added and the layers separated. The aqueous layer was then
extracted with diethyl ether (10 mL × 3), and the combined organic
layers were washed with brine (20 mL) and dried over anhydrous
Na2SO4. It was filtered and concentrated to give the crude residue,
which was purified by Preparative HPLC (40–72% ACN in H2O) to give
product (8b) as a white solid (20 mg, 7.16%). ESI LC/MS: m/z calcd. for
exposure to 200 μM glutamate, which resulted in nearly complete
neuronal death without astrocytic damage (100%).
2.3.3. Measurement of H2O2
A ROS-specific probe, CM-H2DCFDA (1 μM, Invitrogen) and 200 μM
H2O2 were incubated for 30 min and then without or with indicated
concentrations of 8 for additional 1 h. Fluorescent intensity was
measured by Envision multiplate reader at a wavelength of 485/535 nm
(ex/em).
2.3.4. ROS-staining in cells
Primary cortical cells were incubated with 1 μM CM-H2DCFDA in
C
30H33ClO5 [M]+: 509.21; found [M]+: 509.05 and [M + 2]+: 511.0. 1H
growth medium for 30 min and then washed with PBS for three times.
ROS-stained cells were observed under EVOS Cell Imaging Systems
(Thermo Fisher Scientific) at a wave length of excitation 470/20 nm and
emission 510/42 nm.
NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 13.06 (s, 1H, OH), 10.07 (s,
2H, CHO), 7.22 (d, J = 8.68 Hz, 2H), 7.03 (d, J = 6.88 Hz, 2H), 3.47 (d, J
= 11.44 Hz, 1H), 2.14–2.07 (m, 2H), 1.94–1.78 (m, 2H), 1.70–1.57 (m,
3H), 1.51–1.38 (m, 5H), 1.36–1.31 (m, 1H), 1.27–1.24 (m, 1H),
1.09–1.04 (m, 1H), 1.01 (apparent d, 6H), 0.86 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 192.30, 191.58, 169.50, 168.57, 163.42, 141.99, 131.81
(3C), 128.37 (2C), 105.22, 104.64, 104.15, 84.12, 49.94, 47.43, 44.04,
39.87, 37.56, 36.94, 35.44, 35.09, 33.47, 30.61, 29.39, 26.06, 23.87,
20.73, 20.11.
2.3.5. Cell culture
CHO (Chinese hamster ovary), A2780 (Ovary cancer), and BHP
(Thyroid cancer) cell lines were purchased from the American Type
Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured at
37 ◦C in a 5% CO2 humidified incubator and maintained in Roswell Park
Memorial Institute (RPMI 1640, Coring Inc, New York, USA) medium
containing 5 mL penicilin streptomycin, and 10% heat-inactivated fetal
bovine serum (FBS). RAW264.7 murine macrophages of immune cell
lines were purchased from the Korean Cell Line Bank (KCLB®, Seoul,
Korea). Cells were cultured at 37 ◦C in a 5% CO2 humidified incubator
and maintained in high glucose Dulbecco’s Modified Eagle Medium
(DMEM, Coring Inc, New York, USA) medium containing 5 mL penicilin
streptomycin, and 10% heat-inactivated fetal bovine serum (FBS).
2.2.5. Preparation of of bromo (+)-Psiguadial B (8c)
To a stirred solution of diformylphloroglucinol (11, 100 mg, 0.55
mmol) and sodium acetate (4.5 mg, 0.06 mmol, 0.1 equiv.) in acetic acid
(4 mL), 4-bromobenzaldehyde (12c, 203 mg, 1.10 mmol, 2.0 equiv.) and
β -Caryophyllene (5, 0.37 mL, 1.65 mmol, 3.0 equiv.) were added
dropwise at 80 ◦C, the mixture was continuingly stirred for 24 h at 80 ◦C.
Upon cooling to room temperature, brine (5 mL) and diethyl ether (20
mL) were added and the layers separated. The aqueous layer was then
extracted with diethyl ether (10 mL × 3), and the combined organic
layers were washed with brine (20 mL) and dried over anhydrous
Na2SO4. It was filtered and concentrated to give the crude residue,
which was purified by Preparative HPLC (40–72% ACN in H2O) to give
product (8c) as a pale cream solid (22 mg, 7.24%). ESI LC/MS: m/z
calcd. for C30H33BrO5 [M]+: 552.15; found [M]+: 552.95 and [M + 2]+:
554.90. 1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H, OH), 13.06 (s, 1H,
OH), 10.07 (s, 2H, CHO), 7.37 (d, J = 8.6 Hz, 2H), 6.97 (d, J = 6.64 Hz,
2H), 3.46 (d, J = 11.44 Hz, 1H), 2.14–2.06 (m, 2H), 1.93–1.78 (m, 2H),
1.70–1.57 (m, 3H), 1.51–1.38 (m, 5H), 1.36–1.31 (m, 1H), 1.27–1.24
(m, 1H), 1.08–1.04 (m, 1H), 1.01 (apparent d, 6H), 0.86 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 192.30, 191.58, 169.48, 168.57, 163.43,
142.54, 131.31 (2C), 129.56 (2C), 119.84, 105.14, 104.64, 104.15,
84.12, 49.89, 47.43, 44.04, 39.95, 37.56, 36.94, 35.44, 35.09, 33.47,
29.70, 29.39, 26.06, 23.86, 20.73, 20.11.
2.3.6. Cell viability assay
Cells were seeded in a 96-well culture plates and cultured for over-
night and then treated with various concentrations of psiguadial B an-
alogues (compound 8, and 8a-c) for 24 h. Cell viabilities were evaluated
using CellTiter-Glo reagent (Promega. CA, USA) which was added to
each well (20 μ
L) and incubated at 37 ◦C for 10 min. Absorbance were
read using a EnVision Multilabel Reader (Perkin-Elmer, Waltham, MA,
USA) at a luminescence signals.
2.3.7. Production of nitric oxide (NO) and cytokines (TNF-α and IL-6)
Immune cells (1 × 105 cells/well) were seeded in 24-well culture
plate and cultured for 12 h. Cells were pre-treated with various psi-
guadial B analogues (compound 8, and 8a-c) or dexamethasone of 10
μ
M for 1 h and then co-incubated with 500 ng/mL of LPS for 24 h. NO
concentration in medium were determined using a Griess assay. Griess
reagent (50 L) was added to media supernatant (50 L) and then
incubated at 37 ◦C for 15 min in the dark. Absorbance was measured at
520 nm. NO concentrations were caculated using 0–100 M sodium
nitrite standard. TNF- , and IL-6 expression levels in culture medium
were quantified using a sandwich-type ELISA kits.
μ
μ
2.3. General procedure for the evaluation of biological activity
μ
α
2.3.1. Cortical cell cultures
Cortical cell cultures were prepared from embryonic day 14 (E14)
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T. Man Kadayat et al.
Bioorganic Chemistry 113 (2021) 105027
Scheme 2. Our proposed biosynthetic method to psiguadial B using β -caryophyllene.
Fig. 2. Differences in reaction conditions between previously reported work by Cramer and co-workers [18] and our work to synthesize psiguadial B (8)
using NaOAc.
5

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Bioorganic Chemistry 113 (2021) 105027
(14) and phosphorus oxychloride (15). Diformylphloroglucinol (11)
was mixed with three equivalents of β -caryophyllene (5), and two
equivalents of benzaldehyde (12) in a solution of sodium acetate
(NaOAc) and acetic acid at 80 ◦C for 24 h to produce a mixture of psi-
guadial B (8) and guajadial (10). As proposed in Scheme 2, we believe
that ortho-quinone methide (13) obtained from NaOAc-catalyzed
Knoevenagel condensation of 11 and 12, undergoes hetero-Diels-Alder
reaction with tricyclododec-8-ene (5e) derived from acetic acid-
catalyzed isomerization, and subsequent cyclization of
β -car-
yophyllene (5) to form psiguadial B (8).
Compound 8 was separated from the mixture of 10 using preparative
HPLC. We confirmed the structure of prepared psiguadial B (8) by
comparing NMR spectra with previously reported data of natural psi-
guadial B [9] and the synthetic sample [16] (Table S1). Furthermore, we
synthesized fluorine-, chlorine-, and bromine-containing analogues (8a,
8b, and 8c) of psiguadial B by substituting 1′-position phenyl group with
different para-halo-phenyls following the same short synthetic proced-
ure (Scheme 1). The compounds were successfully purified by auto-
preparative HPLC (GX-281 HPLC, Gilson, USA). The structures of the
synthesized compounds 8 and 8a-c were confirmed by NMR spectra (for
details of their synthesis and LC-MS, HPLC data, see the Supplementary
Data). The yields of these compounds were comparable (5.4–7.2%) to
the method reported by Cramer and co-workers [18]. Most importantly,
both our work and Cramer et al.’s work reported reaction using NaOAc
to synthesize psiguadial B. It is important to note that they reported zero
yield, while here we report above 5% yield, and this finding could be
explained as such difference in reaction solvent and condition (Fig. 2).
This synthetic route involved a simple and one-step reaction with
inexpensive starting compounds and reagents allowing to produce target
compounds in large scale for further bioassay and development.
Fig. 3. Protective effect of psiguadial B (8) and its halogenated analogues on
H2O2-induced cell death in mouse cortical cultures. (A) Cortical cultures were
exposed to 150 μM H2O2 in the absence (black bar) or presence of 8 (red bars),
and to 8 alone (blue bars) at indicated concentrations (1–100 μM) for 24 h. Bars
denote the relative amount of lactate dehydrogenase (LDH) release from cell
into medium (*P < 0.001 vs H2O2). (B) LDH release from cultures exposed to
150 μM H2O2 in absence (black bar) or presence of 10 μM of compound 8 (red
bar), 8a (blue bar), 8b (green bar), and 8c (purple bar), and LDH release from
H2O2 untreated cultures by 8 (orange bar), 8a (pink bar), 8b (bright red bar), or
8c (dark pink bar) at indicated concentration for 24 h (*P < 0.001 vs H2O2).
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
3.2. Neuroprotective effect on mouse cortical cells by chelating ROS
As one of the major aims of this study was to evaluate neuro-
protective effect of psiguadial B and its halogenated analogues, mouse
cortical cultures were exposed to 150 μM H2O2 in the absence or pres-
ence of psiguadial B (8) at indicated concentrations (1, 10, 50 or 100
2.3.8. NO reporter gene promoter assay
μ
M) for 24 h. Psiguadial B (8) efficiently reduced H2O2-induced cell
A pNF-κB-Luc plasmid for NF-kB luciferase reporter assay was ob-
tained from Strategene (LaJolla, CA, USA). Expression vectors were
obtained as followed: Flag-IKKa, Flag-IKKb, and Flag-p65 (M. Karin,
University of California San Diego), Myc-NIK and Flag-TNFR (M. Jung,
Georgetown University), HA-TRAF2 andHA-RIP were developed in
reference laboratory. Transfections were performed as described previ-
ously [38,39]. NF-κB-dependent luciferase activity was measured using
IVIS imaging system (perkins elmer, CA, USA).
death in a dose-dependent manner, when it was measured by release of
lactate dehydrogenase (LDH), a quantitative marker for cell death, in
primary cultures of mouse cortex (Fig. 3A). Even compound 8 exhibited
the neuroprotective effect in a concentration-dependent manner, how-
ever, it started to show toxicity from 50 μM. The MTS and CellTiter-Glo
assay to evaluate cytotoxicity of psiguadial B (compound 8) in HepG2
and HCT116 cancer cell lines is shown in Supplementary Figure S1. We
used 10 μM psiguadial B in further experiments. Moreover, as shown in
Fig. 3B, other halogenated psiguadial B analogues (8a, 8b and 8c) also
2.3.9. Statistical analysis
showed similar neuroprotective effect against H2O2-induced cell death
All data are expressed as the mean ± standard deviation from at least
three representative experiments, and statistical significance was
determined using unpaired Student’s t test. P values less than 0.05 were
considered statistically significant.
at 10 μM.
To determine the ROS-chelating effect of compound 8, we monitored
changes in fluorescence of CM-H2DCFDA, H2O2-specific indicator. As
shown in Fig. 4A, compound 8 reduced fluorescent intensity increased
by H2O2 in a concentration-dependent manner. In accordance with these
results, compound 8 dramatically decreased cell death and the level of
ROS in cortical cultures exposed to FeCl2, which is well known as
stimulus for H2O2 generation (Fig. 4B and 4C). These results suggest that
psiguadial B analogues have ability to protect neurons from oxidative
stress in neurodegenerative diseases.
3. Results and discussion
3.1. Short synthetic route of psiguadial B (8) and its halogenated
analogues (8a-c)
As indicated in Scheme 1, our initial aim was to synthesize psiguadial
B (8) using concise synthetic procedure. For this, diformylphlor-
oglucinol (11) was first prepared with previously reported methods
[17,40], by treating phloroglucinol (17) with two equivalent amounts of
Vilsmeier reagent (16) that was obtained from N,N-dimethylformamide
3.3. Anti-inflammatory effects of compounds via inhibition of expression
of nitric oxide (NO), TNF-α and IL-6
The cytotoxicities of compounds 8 and 8a-c on the viability of
6

T. Man Kadayat et al.
Bioorganic Chemistry 113 (2021) 105027
Fig. 4. Inhibitory activity of psiguadial B (8) against reactive oxygen species (ROS)-induced cell death in mouse cortical cultures. (A) To assess H2O2-chelating
efficacy, 1 M CM-H2DCFDA was mixed with 200 M H2O2 for 30 min and then treated without (black bar) or with indicated concentrations of 8 (red bars). The
fluorescent intensity of CM-H2DCFDA was measured by microplate reader at 485/535 nm (ex/em). (B) lactate dehydrogenase (LDH) release from cultures exposed to
10 M FeCl2 in absence (black bar) or presence of 10 M of compound 8 (+Comp 8, red bar), and to 10 M of compound 8 alone (Comp 8, blue bar) for 24 h (*P <
0.001 vs FeCl2). (C) Cultures were exposed to 10 M FeCl2 in absence or presence of 10 M of 8, and to FeCl2 non-treated cells with same concentration of 8 for 24 h.
M CM-H2DCFDA for 30 min and observed under fluorescent
m. (For interpretation of the references to colour in this figure legend, the
μ
μ
μ
μ
μ
μ
μ
Phase contrast images were obtained under light microscope (upper panels). Cells were stained with 1
μ
microscope at excitation 470/22 and emission 510/42 nm (lower panels). Scale bar, 125
reader is referred to the web version of this article.)
μ
various cell lines were evaluated in range from 0.7 to 50
μ
M (Fig. 5)
significantly decreased nitric oxide in immune cells exposed to LPS,
expression of nitric oxide was markedly increased upon exposure to LPS.
However, cotreatment of compounds (DEX, 8, and 8a-c) and LPS
significantly inhibited overnight cotreatment of nitric oxide production.
Similarly, to determine whether these compounds affect the expressions
using normal cells (CHO), immune cells (RAW 264.7), and cancer cells
(A2780 and BHT). Since all the tested compounds were non-toxic at
given concentrations, we used 10 μM in further experiments. Several
studies have reported that the inflammation and oxidative stress are
interlinked to cause neurodegeneration [34,35]. On the basis of the
promising results of neuroprotective effects of compound 8, and 8a-c as
antioxidant in cortical cells, we were interested to further investigate
whether these compounds have ability to protect inflammation. NF-κB,
an important transcriptional activator for inflammatory diseases, is
activated by oxidative stress and pro-inflammatory cytokines such as
of pro-inflammatory cytokines, levels of TNF-
α and IL-6 were assessed in
LPS-induced immune cells. When CHO cells were treated with 10
μ
M
LPS, levels of TNF-α and IL-6 were highly increased. However, com-
pounds 8 and 8a-c significantly inhibited the expressions of both TNF-
α
and IL-6 in LPS-induced immune cells (Fig. 6B and 6C).
TNF-
α and IL-6. Thus, we investigated anti-inflammatory activity of
3.4. Effect of compounds on NF-κB activation
these compounds via the production of nitric oxide (NO), TNF-α and IL-6
by suppressing the NF-κB pathway in lipopolysaccharide (LPS)-induced
immune cells. We performed NO assay, cytokine assays, and NF-κB
promoter assay in CHO cells, with dexamethasone (DEX) as a reference
compound (Fig. 6).
NF-κB is an important transcriptional activator for inflammatory
diseases. Luciferase assay with bioluminescence imaging was performed
to understand the mechanism underlying the inhibitory activity of
compounds on LPS-induced expressions of NO, TNF-α and IL-6 in im-
Fig. 6A illustrates that addition of compounds (DEX, 8, and 8a-c)
mune cells. Compound 8, and 8a-c inhibited the level of luciferase
7

T. Man Kadayat et al.
Bioorganic Chemistry 113 (2021) 105027
Fig. 5. Effect of psiguadial B (8) and its halogenated analogues (8a-c) on cell viability in various cell lines. Cell proliferation in CHO: normal cells, RAW 264.7:
immune cells, A2780 and BHT: cancer cells with or without addition of compounds 8 and 8a-c.
signal, indicating that these compounds downregulate NF-κB activity in
LPS-induced immune cells (Fig. 6D). These results suggested that psi-
guadial B and its halogenated analogues have potential to be used as
neuroprotective agents by inhibiting production of nitric oxide and
expression of pro-inflammatory mediators via suppression of NF-κB
activity.
Furthermore, inhibition of LPS-induced expressions of NO, TNF-α and
IL-6 in immune cell by suppression of NF-κB pathway suggested that
these compounds have anti-inflammatory activity. These findings pro-
vide evidence for the development of psiguadial B-based neuro-
protective and anti-inflammatory drugs against brain diseases and
immunological disorders. Further research on synthesis and neuro-
protective effects of psiguadial B derivatives with a modified diformyl-
phloroglucinol group to identify lead compounds is ongoing and
planned to be reported in near future.
4. Conclusions
In conclusion, we developed a short synthetic route of psiguadial B
(8) and its halogenated analogues (8a-c) by coupling β -caryophyllene,
benzaldehyde and diformylphloroglucinol using a non–toxic and
cheaper sodium acetate catalyzed single step approach. The preliminary
in vitro study in H2O2-induced cell death using primary cultures of mouse
cortical neurons demonstrates psiguadial B and its halogenated ana-
logues as novel and effective neuroprotective agents by chelating ROS.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
8

T. Man Kadayat et al.
Bioorganic Chemistry 113 (2021) 105027
Fig. 6. Effect of psiguadial B and its halogenated analogues in immune cells. (A) Effect of compounds on LPS-induced NO production in immune cells. Immune cells
were exposed to 1 μg/mL LPS and combination of 10 μM dexamethasone (DEX), compound 8, and 8a-c. (B-C) Effect of compounds on expressions of TNF-α (B) and
IL-6 (C) in medium as determined by enzyme-linked immunosorbent assay (ELISA) in LPS-induced immune cells. Immune cells were pre-treated with indicated
concentration of DEX, compound 8, and 8a-c for 1 h and then with LPS (500 ng/mL, 24 h). (D) Effect of compounds on NF-κB activation in LPS-induced immune cells
as determined by luciferase assay with IVIS imaging system (**P < 0.01, ***P < 0.001).
[6] S. Begum, S.I. Hassan, B.S. Siddiqui, F. Shaheen, M.N. Ghayur, A.H. Gilani,
Acknowledgements
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This work was supported by the National Research Foundation of
Korea (NRF) grants funded by the Korean Government Ministry of Sci-
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NRF-2017R1D1A1B03030196, NRF-2018R1D1A1B07047417, NRF-
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2021M3A9G101647531,
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Appendix A. Supplementary material
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