Anticandidal formyl phloroglucinol meroterpenoids: Biomimetic synthesis and in vitro evaluation

Article abstract of DOI:10.1016/j.bioorg.2020.104248

Inspired by the diversity-oriented synthesis, some novel formyl phloroglucinol meroterpenoids were synthesized via biomimetic synthesis using essential oils. Eight of them were demonstrated with good in vitro fungicidal activity against Candida albicans a

Full text of DOI:10.1016/j.bioorg.2020.104248

Bioorganic Chemistry 104 (2020) 104248
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Anticandidal formyl phloroglucinol meroterpenoids: Biomimetic synthesis
and in vitro evaluation
⁎
Lin–Fang Zhong, Zhi–Chun Shang, Fu–Juan Sun, Pan–Hu Zhu, Yong Yin, Ling–Yi Kong ,
⁎
Ming–Hua Yang
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China
Pharmaceutical University, Nanjing 210009, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Inspired by the diversity-oriented synthesis, some novel formyl phloroglucinol meroterpenoids were synthesized
via biomimetic synthesis using essential oils. Eight of them were demonstrated with good in vitro fungicidal
activity against Candida albicans and C. glabrata. Compound c2 showed the best anticandidal ability that was
powerfully comparable to fluconazole when testing against several strains in vitro. The antibiofilm activity was
also found for the c2 treating group which was evidenced to block the hyphal elongation and filamentation of C.
albicans. Therefore, compound c2 is a promising candidate for further antifungal-based structure modification.
Formyl phloroglucinol meroterpenoids
Essential oil
Anticandidal activity
Antibiofilm activity
1
. Introduction
Natural products have long been considered a potential source of
obvious anticandidal activity to FPMs, although more than ten types
with different hybrid patterns were found. The terpenoid involved in
forming FPMs undoubtedly is one of the key elements concerning their
antifungal ability. If we were able to screen FPMs with more different
terpenes, it was possible to find more effective candidates.
novel drug leads on all sides. The development of antifungals benefits a
lot from nature too [1–2]. The echinocandin is the product of Aspergillus
nidulans and the amphotericin B and nystatin (polyenes) are naturally
occurring polyethylenes in different Streptomyces species [3–6]. How-
ever, there are relatively a few classes of antifungals used in clinic and
most of them have been using for decades [7–8]. Their widespread and
prolonged application leads to the increasingly severe drug resistance
problem in clinic [9–10]. Developing drugs with novel antifungal tar-
gets is one of most useful solutions. Natural products with various and
novel structure are still wonderful candidate pool, and some molecules
with novel targets like caspofungin and celastrol already shown their
perfect application prospects in recent researches [11].
Terpenoids were found to be potential efflux pump substrates of
fungi, which was evidenced by the fact that they were active against
efflux pump-deficient C. albicans but inert towards wild-type strains
[20]. It means they cannot be intracellularly retained enough to inhibit
or kill general fungal strains. Structural modification employing orga-
nelle-targeting groups was proved to be a useful way to enhance or
restore the drug activity [21–22]. Several terpenoids were restored
their anticandidal activity by conjugating them to the mitochondria-
targeting triphenylphosphonium cation [20]. The endoplasmic re-
ticulum-localized azole was also reported to remarkably improve anti-
fungal activity against a panel of Candida spp. [23]. In light of above
findings, we posited that the phloroglucinol group in FPMs could have
exerted a retaining effect to terpene group too.
Formyl phloroglucinol meroterpenoids (FPMs) are characteristic
metabolites of the genus Eucalyptus. They are the phloroglucinol hy-
brids of terpene moieties and structurally diverse in the terpenoid types
plus conjugate patterns [12–14]. Our previous research on the leaves of
Eucalyptus robusta Smith was resulted in a lot of FPMs with potential
anticandidal activity against different Candida spp., including flucona-
zole-resistant strains [15–17]. Some of them exerted a strong inhibitory
effect against C. albicans biofilms, and some showed synergistic effect
with azoles [18–19]. These qualities make FPMs potential candidates
for antifungal agents. Analyzing the anticandidal structure-activity re-
lationships, we found that only three terpene types could provide
More FPMs were desired to discover effective FPMs and to verify our
function hypothesis on phloroglucinol group. Different routes had been
reported for biomimetic synthesis of FPMs [14,24], which therefore
could be an efficient option for us. However, it would be difficult to
gather many terpenoids as reagents, and the bioassay screening of
abundant products also could be a heavy task. Inspired by the diversity-
oriented synthesis [25–27], essential oils with rich terpenes con-
stituents could be ideal reaction reagents. It is because of that different
⁎
Corresponding author.
E-mail addresses: cpu_lykong@126.com (L. Kong), skeepjack@163.com (M. Yang).
https://doi.org/10.1016/j.bioorg.2020.104248
Received 1 March 2020; Received in revised form 15 May 2020; Accepted 28 August 2020
Available online 01 September 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.

L. Zhong, et al.
Bioorganic Chemistry 104 (2020) 104248
terpenes in them would generate diverse FPMs in one reaction, which
combined with bioactivity-guided isolation would improve our effi-
ciency of finding more antifungal FPMs. Hence, we conducted this
synthetic strategy by using commercially available essential oil and
successfully obtained several novel anticandidal FPMs.
Table 1
In vitro anticandidal activity of the best fractions of products derived from 12
essential oils respectively.
C. albicansa
C. glabrataa
Fractions
Cedar oil’s product
54.90 ± 4.21
129.50 ± 1.23
> 500
80.50 ± 3.26
138.40 ± 2.71
196.50 ± 1.21
210.20 ± 2.10
137.50 ± 1.24
74.80 ± 1.52
230.60 ± 2.14
> 500 ± 1.52
190.80 ± 1.71
182.10 ± 2.12
149.30 ± 3.10
125.40 ± 1.31
1.00 ± 0.23
Spikenard oil’s product
Zedoary oil’s product
Citronella’s product
2
. Results and discussion
> 500
Atractylis oil’s product
Ginger oil’s product
141.60 ± 5.81
60.30 ± 3.14
> 500
2.1. Chemistry
Chamomile oil’s product
Orange flower oil’s product
Rosemary oil’s product
Lavender oil’s product
Clove oil’s product
2
.1.1. Biomimetic synthesis of FPMs
211.40 ± 1.51
123.50 ± 2.71
194.10 ± 0.93
162.70 ± 4.72
130.10 ± 1.30
0.50 ± 0.13
The phloroglucinol-terpene adducts are biosynthetically hypothe-
sized to arise from the Diels-Alder (DA) cycloaddition [14,24]. Our
previous study had proved that biomimetic synthesis conducting DA
conditions was an effective way to produce them [28–30]. Meanwhile,
the previous antifungal structure-activity relationships indicated that
rather than the single CeC bonds between two moieties, the formation
of oxygen heterocycle which is the typical product of DA reaction could
contribute to antifungal activity [15–17]. The hetero DA reaction is
therefore an appropriate way in this case.
Peppermint oil’s product
b
Fluconazole
C. albicans (SC5314); C. glabrata (ATCC 15126).
a
b
MIC50 value; μg/mL.
Positive drug.
6
, from H-3 to C-2/C-5, and from H
2
-4 to C-2/C-6 revealed a menthane-
Since the number of terpenes in essential oil is directly related to the
variety of FPMs it would produce, we selected 12 essential oils that are
different in terpenes as reaction reagents. They were listed in Table S1
along with their plant source. As shown in Table S2, several conditions
were investigated to promote the DA reaction. The reaction with the use
of sodium acetate (10 equiv) in acetic acid at 100 °C for 16 h obtained a
better yield of FPMs’ mixture which also showed numbers of peaks in
HPLC analysis. This condition was accordingly applied, and the syn-
thetic route was illustrated in Scheme 1.
1
(6)
type monoterpene with the Δ
double bond. The downfield-shifted C-
7
(δ
C
82.7) and the HMBC correlation from H -7′ to C-7/C-8/C-3′/C-1′
2
confirmed that the two moieties were fused through a dihydropyran
ring. Obviously, compound c1 is the adduct of formyl-phloroglucinol
7
(8)
and limonene, and a DA addition was happened on the Δ
double
bond of limonene. Compound c2 was determined as a C-7 epimer of c1.
It had a same molecular formula and very similar 1D and 2D NMR data
to c1, but the Me-9 had a ROESY correlation with H-4. This was due to
the achiral condition using for the synthesis above.
The 12 essential oils respectively produced 12 crude mixtures of
FPMs by conducting the route, and every product was further divided
into 4 fractions by silica gel column chromatography. The 48 fractions
were then preliminarily submitted to the disk diffusion assay and the
anticandidal fractions were double checked by broth microdilution
assay. As shown in Table 1, fractions of products derived from cedar oil
and ginger oil had the best antifungal activity against the two main
pathogens of candidiasis. Their abilities were confirmed by disk diffu-
sion assay (Fig. 1), in which both fractions showed obvious con-
centration-dependent inhibition zones. Thus, these two fractions were
further purified by chromatography which led to the isolation of an-
ticandidal c1-c5 and g1-g3 (Fig. 2) along with the inactive compounds
c6 and g4-g7 (Structures in Fig. S1, and NMR data in Tables S3 and S4).
c1-c6 were the product of cedar oil and g1-g7 were isolated from the
product of ginger oil. All the isolates were the [4 + 2] cycloaddition
products of the conjugated diene from formyl-phloroglucinol and the
dienophile from terpenes. The formation of the c1-c5 and g1-g3 were
described in scheme 2.
Compounds c3 and c4 were a pair of epimer, since they had the
same molecular formula and almost identical NMR data. The HMBC
correlations clearly (Fig. S2) revealed that they were also the adducts of
1
(6)
limonene, but the Δ
double bond was involved in fusing with the
formyl-phloroglucinol in c3 and c4. The ROESY correlations between
H-1/Me-10, H-5α/Me-10, H-3/H-2β, H-3/H-5β and H-1/H-2α were
found in c3, while c4 had ROESY correlations between Me-10/H-1 and
H-3/H-1. Thus, c3 and c4 were C-9 epimers.
The molecular formula C19
H
22
5
O of both c5 and g1 revealed that
these two synthetic FPMs were a pair of isomers, and the characteristic
upfield-shifted H-3 indicated that they were the formyl-phloroglucinol
hybrids with the sabinene [31] and its isomers. This was confirmed by
the HMBC correlations which also determined the different linkage of
two moiety in them (Fig. S2). The HMBC correlations from H
2
-7′ to C-6,
-2 to C-7, Me-8 to C-3/C-9 and
Me-9 to C-3 along with the chemical shift of C-6 (δ 91.2) determined
the oxa-spiro [5.5] ring in c5 as the coupling pattern of euglobal Ic
32]. The HMBC correlations from H -7′ to C-4/C-5/C-6 revealed that
H
2
-10 to C-1, H
2
-4 to C-6, H
2
-5 to C-3, H
2
C
[
2
C-5 and C-6 in g1 were participated in forming the dihydropyran ring
with formyl-phloroglucinol. In addition, their relative configurations
were determined by the ROESY correlations shown in Fig. S2.
Compounds g2 and g3 were determined as the adducts with pinene-
type monoterpene, since their NMR data were similar to those of ro-
bustadial A [33–34]. The HMBC correlations (Fig. S2) further de-
termined that the double bond of an α-pinene formed the dihydropyran
with phloroglucinol in g2, and the double bond of a β-pinene formed
the oxa-spiro [5.5] ring in g3.
2
.1.2. Structure determination of bioactive FPMs
Compound c1 was determined to have the molecular formula
1
C
19
H
22
O
5
by HRESIMS. Its H NMR data clearly showed the char-
acteristic signals of formyl-phloroglucinol which were the two hy-
drogen-bonded phenolic hydroxyl groups (δ 13.44 and 13.25, each
H, s) and the two formyl groups (δ 10.05 and 10.16, each 1H, s).
H
1
H
Aside from those of above moiety and a carbon came from the for-
1
3
maldehyde in reaction, there were 10 carbons left in the C NMR
spectrum, which revealed a monoterpene moiety for c1. The HMBC
correlations (Fig. S2) from Me-9 to C-3/C-8, from Me-10 to C-1/C-5/C-
The Snatzke helicity rules [15] were used to determine the absolute
Scheme 1. The synthetic route for FPMs. Reagents
CHO
OH
CHO
OH
and conditions. (i) DMF, POCl
3
, Dioxane, 12 h, rt;
HO
OH
(iii)
HO
O
(
i)
(
ii) H
2
O, 4 h, 0 °C; (iii) Essential oil, CH
3
COOH,
(
Terpene
)
CH
3
COONa, HCHO, 16 h, 100 °C.
(ii)
OHC
OHC
HO
OH
OH
2

L. Zhong, et al.
Bioorganic Chemistry 104 (2020) 104248
Fig. 1. The anti-candida albicans (SC5314) effect of crude fractions in disk diffusion testing. (a) The inhibition zones of fluconazole; (b) The inhibition zones of the
best active fraction of cedar oil’s product; (c) The inhibition zones of the best active fraction of ginger oil’s product.
configuration of above eight compounds. As shown in Fig. S3, each
helical direction of dihydropyran rings was predicted according to the
signs of the Cotton effects around 280 nm, and therefore those chiral
centers on the dihydropyran rings were determined as shown.
Candida strains especially to C. krusei was comparable to fluconazole.
Six clinical isolates were also applied in this study. As shown in Table 3,
compounds c1-c4 all showed fluconazole-comparable activity to flu-
conazole sensitive strains but inert towards fluconazole resistant iso-
lates. There are multi-aspect reasons for azoles resistance. Over-
expression or point mutation of ERG11 gene and increase of efflux
pumps like Cdr1p and Mdr1p are the most frequently cause find in
Candida spp. [9,20]. The overexpression of efflux pump was relatively
more possible to be response for the inert of c1-c4, since the efflux
pumps with broad substrates could significantly decrease their in-
tracellular concentration. The two fluconazole-resistant strains (S381
and CA422 in Table 3) were thereafter quantified for their expression of
CDR1 and CDR2 genes which were more than 10 times overexpressed
when comparing to the reference one (data not shown), thus explaining
the inactive of c1-c4.
2.2. Biological evaluation
2
.2.1. In vitro anticandidal activity
All the 13 compounds were evaluated for their in vitro anticandidal
activity on C. albicans and C. glabrata. As shown in Table 2, eight
compounds showed certain activity. They are all the phloroglucinol
adducts of monoterpenes, and these monoterpene precursors were
happened to be reported for their antifungal activities before [35]. For
example, limonene was reported to inhibit Candida albicans growth by
inducing apoptosis [36]. However, it is noticed that the limonene ad-
ducts, compounds c1-c4, were much more effective, which could be the
contribution from formyl-phloroglucinol. Compounds c1-c4 had a re-
latively notable influence and thus were submitted to further evalua-
tion. Another four Candida species, namely C. parapsilosis, C. tropicalis,
C. guilliermondii and C. krusei were used to evaluate the comprehensive
anticandidal ability, and they showed similar drug susceptibility like C.
albicans and C. glabrata (Table 3). Their activities against different
2.2.2. The cytotoxicity assessment
Fungi is eukaryote like mammalian cells, which means side effects
might be caused by antifungal agents. It is thus necessary to consider
the toxicity of c1-c5 and g1-g3. Human normal liver cell line LO2 was
used for the in vitro cytotoxicity evaluation. All the eight compounds
did not display considerable toxicity at 100 µg/mL which demonstrated
1
10
CHO
CHO 10
5
8 'C HO
9
HO
O
HO
O
3
HO
O
7'
5'
3'
7
H
1
H
OHC
OHC
3
OHC
7
H
1
'
9'
OH
c2
OH
OH
8
c3
c1
CHO
7
CHO
CHO 10
O
1
HO
O
3
HO
O
HO
OHC
1
5
OHC
3
OHC
H
H
OH
c4
OH
c5
OH
g1
3
CHO
7
1
CHO
HO
O
HO
O
1
5
8
8
5
OHC
7
OHC
H 3
OH
g2
OH
g3
Fig. 2. Structures of compounds c1-c5 and g1-g3.
3

L. Zhong, et al.
Bioorganic Chemistry 104 (2020) 104248
CHO
OH
HO
OH
OHC
c1: 7S
c2: 7R
c3: 1S, 6S
c4: 1R, 6R
CHO
O
CHO
OH
HO
OHC
HO
O
OHC
[
4+2]
OH
[
4+2]
CHO
OH
CHO
OH
CHO
OH
HO
OHC
O
[
4+2]
HO
O
[4+2]
HO
O
c5
g3
OHC
OHC
[
4+2]
[
4+2]
CHO
CHO
HO
O
HO
OHC
O
OHC
OH
OH
g2
g1
Scheme 2. The formation of compounds c1-c5 and g1-g3.
Table 2
antibiofilm effect started from the biofilm initiation stage. In another
assay using mature biofilm, c2 was also found to effectively eliminate
the mature biofilms that had grown for 24 h in a dose dependent way.
When treated with 100 μg/mL c2, almost all mature biofilms (90%)
were removed. Above experimental results revealed that compound c2
could prevent C. albicans biofilm formation and disrupt mature biofilm
effectivity.
In vitro anticandidal activity of the compounds c1-c5 and g1-g3.
C. albicansa
C. glabrataa
Compounds
c1
16.97 ± 1.16
8.65 ± 1.33
> 50
10.17 ± 1.09
13.51 ± 2.20
20.27 ± 1.09
22.78 ± 4.52
21.40 ± 3.17
> 50
c2
c3
c4
17.17 ± 2.60
> 50
c5
g1
21.64 ± 4.07
23.19 ± 3.45
> 50
2
.2.4. Observating the c2 treated biofilms under microscopy
g2
> 50
To provide visualized evidence, the antibiofilm effect was further
g3
28.74 ± 4.92
1.00 ± 0.10
b
Fluconazole
0.50 ± 0.24
confirmed by the analysis of confocal laser scanning microscopy
(
CLSM) and scanning electron microscopy (SEM). As shown in Figs. 4a
C. albicans (SC5314); C. glabrata (ATCC 15126).
and 5a to c, the long filaments developed into an expanding network in
the control group. After the treatment of c2, a disruption was found and
enhanced in a dose-dependent manner. In the CLSM results visualized
by fluorescein diacetate, the dead hyphae labeled in red was sig-
nificantly increased at the concentration of 25 μg/mL, and a large
proportion of dead hyphae was found in the treating group of 50 μg/mL
c2. These results clearly demonstrated that the viability of hypha re-
duced as c2 increased (Fig. 4c and d). In the SEM analysis, we found
that the biofilm of control group (Fig. 5a–c) developed long and tightly
twisted filaments and the cells density were large. In the c2 treatment
group, cells were found being restricted at the budding yeast stage and
the density of biofilm cells was consequently reduced (Fig. 5k and l).
When increasing c2, the hypha decreased and yeast increased. When
the c2 concentration increased to 50 μg/mL, few long hyphae were
seen, which indicated that c2 could block hyphal elongation and fila-
mentation. These results demonstrated that c2 can reduce cell viability
and it inhibited biofilm by locking C. albicans in the yeast form of the
growth.
a
b
MIC50 value; μg/mL.
Positive drug.
these compounds are safety at their active concentration ranges (Fig.
S4).
2
.2.3. The in vitro C. albicans biofilms inhibition of c2
Biofilms are a protected niche for microorganisms, where they are
inherently tolerant to antimicrobial treatment. The current antifungal
agents are low in susceptibility of Candida biofilms [37–38]. Given the
severity of biofilm-related diseases, antibiofilm activity is an important
capability for potential antifungal candidates. A series of bioassays were
therefore carried out to evaluate the biofilm inhibition of c2. The
crystal violet (CV) method was used to see if c2 could prevent biofilm
formation. As shown in Fig. 3, about 50% and 66% suppression were
obtained at the concentrations of 12.5 and 25 μg/mL respectively. The
biofilm growth was obviously disrupted upon c2 treatment and the
4

L. Zhong, et al.
Bioorganic Chemistry 104 (2020) 104248
Table 3
In vitro anticandidal activity of the compounds c1-c4.
Strains
c1
c2
c3
c4
Fluconazoleb
a
C. parapsilosis
6.12 ± 1.21
11.90 ± 2.41
4.56 ± 0.32
7.16 ± 1.73
4.72 ± 3.87
> 50
4.80 ± 3.24
5.20 ± 1.47
9.89 ± 2.74
5.32 ± 1.72
2.94 ± 0.15
22.05 ± 0.23
5.74 ± 1.31
9.13 ± 2.52
> 50
7.07 ± 1.83
19.07 ± 4.32
9.93 ± 2.74
7.55 ± 1.92
12.93 ± 2.75
> 50
12.41 ± 3.91
12.30 ± 3.71
4.78 ± 1.02
7.59 ± 2.95
10.12 ± 3.72
> 50
1.60 ± 0.92
0.90 ± 0.01
2.50 ± 0.81
8.00 ± 2.78
0.50 ± 1.89
30.00 ± 0.03
1.00 ± 0.06
1.00 ± 0.11
> 50
a
C.tropicalis
a
C. guilliermondii
a
a
a
C.krusei
YEM12
YEM14
a
a
a
CA21
10.02 ± 4.21
10.68 ± 3.21
> 50
4.45 ± 1.67
8.88 ± 2.86
> 50
12.41 ± 4.72
12.30 ± 4.21
> 50
CA13
S381
CA422a
> 50
> 50
> 50
> 50
> 50
C. parapsilosis (ATCC 22019); C. tropicalis (ATCC 750); C. guilliermondii (ATCC 6260); C. krusei (ATCC 1182).
YEM12, YEM14, CA21, CA13, S381 and CA422 are clinical isolates of C. albicans.
a
b
MIC50 value; μg/mL.
Positive drug.
Fig. 3. C2 inhibits C. albicans biofilms in vitro. (a) Effects of different concentrations of c2 on mature biofilm. (b) Effects of different concentrations of c2 on biofilm
formation. Bars represent means ± SDs from three experiments. *, P < 0.05.
3
. Conclusion
instrument with a shim-pack RP-C18 (20 × 200 mm). Nuclear magnetic
1 13
resonance ( H NMR and C NMR) spectra were recorded on a Bruker
AVIII-500 and Bruker AVIII-600 spectrometer with TMS as an internal
standard.
Enlightened by the diversity-oriented synthesis, commercially
available essential oils that are abundant in terpenes were applied as
reagents in the biomimetic synthesis of FPMs, which generated various
FPMs in one reaction. The following antifungal bioassay-guided isola-
tion successfully led to eight anticandidal FPMs. Compound c2, one
limonene-derived FPM, is the most active one. It has a broad-spectrum
anticandidal ability which was powerfully comparable to fluconazole
when testing against several Candida strains in vitro. Compound c2 also
possesses the antibiofilm activity which was evidenced by blocking
hyphal elongation and filamentation. These qualities make c2 a pro-
mising candidate for further antifungal-based structure modification.
4.1.1. General procedure for the synthesis of 2,4-diformyl phloroglucinol
2,4-diformyl phloroglucinol was synthesized as described in the
literature [24].
Phosphoryl chloride (8 mL, 83.5 mmol) was added dropwise to DMF
(6.5 mL, 83.5 mmol) with strong stirring, at room temperature under a
nitrogen atmosphere. The mixture was stirred for 30 min. This
Vilsmeier reagent was then slowly added to a stirred solution of an-
hydrous phloroglucinol (5 g, 39.5 mmol) in dioxane (25 mL) at ambient
temperature, under a nitrogen atmosphere. This reaction mixture was
stirred further for 12 h, and it turned into a yellow solid. This solid
mixture was cooled to 0 °C before being added to ice-water slurry
(~200 mL), and stirring was continued for a further 4 h, during which
time a salmon colored precipitate formed. This crude product was then
filtered off and purified by silica gel column chromatography using
petroleum ether/ethyl acetate (3:1) as eluent to get 2,4-diformyl
4
. Experimental section
4.1. Chemistry
Reagents and solvents were purchased from common commercial
suppliers and were used without further purification. Progress of the
reaction was monitored by TLC using silica gel plates with fluorescence
GF254 (Qingdao Haiyang Chemical Plant, Qingdao, China) and the
compounds on the TLC plates were detected under UV light. Column
chromatography was performed on silica gel (200–300 mesh). Optical
rotations were measured with a JASCO P-1020 spectropolarimeter. The
ECD spectra was recorded using a JASCO 810 spectropolarimeter. The
HRESIMS spectra was measured using an Agilent UPLC-Q-TOF analyser.
Analytical HPLC was performed using an Agilent 1260 Series instru-
ment with a DAD detector and shim-pack VP-ODS (250 × 4.6 mm).
Preparative HPLC was performed using a Shimadzu LC-6AD Series
1
phloroglucinol (5.9 g); cream colored solid; yield 82%. H NMR
(DMSO‑d , 500 MHz) δ ppm 5.91 (s, 1H, Ar-H), 10.02 (s, 2H, CHO),
6
12.51 (br s., 2H, OH), 13.53 (br s., 1H, OH).
4.1.2. General procedure for the synthesis of compounds (c1-c6, g1-g7)
A mixture of 2,4-diformyl phloroglucinol (1 g, 5.5 mmol), for-
maldehyde (1.1 mL, 11 mmol), essential oil (3.75 mL, 16.5 mmol) and
sodium acetate (4.5 g, 55 mmol) in acetic acid (30 mL) was stirred
under 100 °C for 16 h. Then, the reaction mixture was cooled to room
temperature. The saturated sodium bicarbonate solution (20 mL) was
5

L. Zhong, et al.
Bioorganic Chemistry 104 (2020) 104248
Fig. 4. Confocal laser scanning microscopy images
of C. albicans biofilms developed in vitro. The
healthy cells with an intact membrane were stained
with fluorescent green and the cells with damaged
membranes stained fluorescent red. (a) Control
group; (b) 12.5 μg/mL c2; (c) 25 μg/mL c2; (d)
5
0 μg/mL c2. (For interpretation of the references
to colour in this figure legend, the reader is referred
to the web version of this article.)
added to neutralize excess acid. The mixture was extracted with CH
2
Cl
2
23.5 (C-8), 23.4 (C-10), 20.2 (C-9), 14.7 (C-7′). HRESIMS m/z 329.1397
-
(
(
3 × 10 mL), and the combined organic layers were washed with brine
[M−H] (calcd for C19
22
H O5, 329.1394).
2
5
20 mL) and dried over Na
2
SO
4
. Filtration and removal of the solvent
Cedartriol B (c2): White power. 34 mg.[ ]
+ 18.2 (c 0.09, MeOH).
D
1
under reduced pressure afforded a yellow oil. Then, the 12 crude pro-
ducts were separated by silica gel chromatography with petroleum
ether/ethyl acetate (100:1 to 1:1, v/v) and divided into 4 fractions
which were then submitted to anticandidal activity assay. The best
anticandidal activity fractions (fraction A from cedar oil’s product and
fraction B from ginger oil’s product) were subject to chromatographic
separation by a reversed-phase C18 column to obtain fractions A1-A2
H NMR (500 MHz, CDCl ) δ ppm 13.44 (s, 1H, 1′-OH), 13.25 (s, 1H, 5′-
3
OH), 10.15 (s, 1H, H-9′), 10.03 (s, 1H, H-8′), 5.04 (br d, J = 3.7, 1H, H-
1), 2.63 (dt, J = 16.7, 6.0 Hz, 1H, H-7′), 2.51 (m, 1H, H-7), 2.08 (m,
1H, H-5), 2.03 (m, 1H, H-2), 2.01 (m, 1H, H-8), 1.94 (m, 1H, H-4), 1.94
(m, 1H, H-5), 1.85 (m, 1H, H-3), 1.85 (m, 1H, H-8), 1.81 (m, 1H, H-4),
1
3
1.66 (s, 3H, H-10), 1.36 (m, 1H, H-2), 1.29 (s, 3H, H-9). C NMR
(125 MHz, CDCl ) δ ppm 192.4 (C-8′), 192.0 (C-9′), 168.8 (C-1′), 168.5
3
(
MeOH/H
2
O, 60:40/100:0, v/v) and fractions B1-B3 (MeOH/H
2
O,
(C-3′), 163.5 (C-5′), 134.0 (C-6), 120.1 (C-1), 104.4 (C-6′), 103.5 (C-4′),
5
5:45/100:0, v/v). Fraction A1 was purified using preparative HPLC
with 87% acetonitrile in water to obtain compounds c1 (23 mg), c2
34 mg), c6 (46 mg). Fraction A2 was separated by HPLC using the
mobile phase MeCN/H O (90:10, v/v) to give compounds c3 (33 mg),
c4 (38 mg), c5 (13 mg). Fraction B1 was subjected to preparative HPLC
100.1 (C-2′), 81.0 (C-7), 42.0 (C-3), 30.6 (C-2), 27.8 (C-4), 26.7 (C-5),
23.9 (C-8), 23.9 (C-10), 22.0 (C-9), 15.0 (C-7′). HRESIMS m/z 329.1392
-
(
[M−H] (calcd for C19
H O5, 329.1394).
22
2
5
Cedartriol C (c3): White power. 33 mg.[ ] + 34.8 (c 0.09, MeOH).
2
D
1
H NMR (500 MHz, CDCl ) δ ppm 13.46 (s, 1H, 1′-OH), 13.27 (s, 1H, 5′-
3
OH), 10.15 (s, 1H, H-9′), 10.06 (s, 1H, H-8′), 4.86 (br s, 1H, H-8), 4.80
br s, 1H, H-8), 2.65 (dd, J = 16.9, 6.3 Hz, 1H, H-7), 2.47 (dd,
J = 16.9, 7.0 Hz, 1H, H-7), 2.34 (br s, 1H, H-3), 2.02 (m, 1H, H-1), 1.85
(
MeCN/H O, 85:15, v/v) to obtain compounds g1 (14 mg), g2 (37 mg),
2
(
g3 (19 mg). Fraction B2 was purified by preparative HPLC with 78%
acetonitrile in water to obtain compounds g4 (42 mg) and g6 (39 mg).
(
m, 1H, H-2), 1.85 (m, 1H, H-4), 1.81 (m, 1H, H-5), 1.78 (m, 1H, H-4),
Fraction B3 was further separated by HPLC (MeCN/H O, 70:30, v/v)
2
1
.75 (s, 3H, H-9), 1.67 (m, 1H, H-2), 1.67 (m, 1H, H-5), 1.43 (s, 3H, H-
affording compounds g5 (26 mg) and g7 (31 mg).
2
5
13
Cedartriol A (c1): White power. 23 mg.[ ] + 15.5 (c 0.11, MeOH).
10). C NMR (125 MHz, CDCl
3
) δ ppm 192.2 (C-8′), 191.7 (C-9′), 169.2
D
1
H NMR (500 MHz, CDCl
3
) δ ppm 13.44 (s, 1H, 1′-OH) 13.25 (s, 1H, 5′-
(C-3′), 168.3 (C-1′), 162.9 (C-5′), 147.3 (C-7), 110.4 (C-8), 104.1 (C-6′),
103.9 (C-4′), 99.5 (C-2′), 80.8 (C-6), 38.0 (C-3), 33.1 (C-4), 32.8 (C-1),
OH) ,10.16 (s, 1H, H-9′), 10.05 (s, 1H, H-8′), 5.38 (dd, J = 4.1, 1.0 Hz,
H, H-1), 2.60 (dt, J = 16.8, 6.3 Hz, 1H, H-7′), 2.52 (m, 1H, H-7′), 2.07
m, 1H, H-5), 2.00 (m, 1H, H-2), 1.98 (m, 1H, H-8), 1.93 (m, 1H, H-4),
.89 (m, 1H, H-5), 1.86 (m, 1H, H-3), 1.85 (m, 1H, H-8), 1.80 (m, 1H,
1
31.8 (C-2), 26.3 (C-5), 25.7 (C-10), 21.9 (C-9), 20.6 (C-7′). HRESIMS m/
-
(
z 329.1396 [M−H] (calcd for C19
H
22
O
5, 329.1394).
2
5
Cedartriol D (c4): White power. 38 mg. [ ] + 30.3 (c 0.09,
1
D
1
1
3
MeOH). H NMR (500 MHz, CDCl
3
) δ ppm 13.45 (s, 1H, 1′-OH), 13.26
H-4), 1.65 (s, 3H, H-10), 1.30 (s, 3H, H-9), 1.26 (m, 1H, H-2). C NMR
(
s, 1H, 5′-OH), 10.15 (s, 1H, H-9′), 10.07 (s, 1H, H-8′), 4.67 (br s, 1H, H-
(125 MHz, CDCl
C-1′), 163.3 (C-5′), 134.7 (C-6), 119.7 (C-1), 104.4 (C-6′), 103.8 (C-4′),
00.6 (C-2′), 82.7 (C-7), 41.6 (C-3), 30.7 (C-2), 27.4 (C-4), 26.6 (C-5),
3
) δ ppm 192.1 (C-8′), 191.8 (C-9′), 169.0 (C-3′), 168.3
8
), 4.64 (br s, 1H, H-8), 2.73 (dd, J = 16.9, 6.5 Hz, 1H, H-7′), 2.42 (br
(
d, J = 16.9, 1H, H-7′), 2.15 (m, 1H, H-5), 2.02 (m, 1H, H-3), 1.91 (m,
1
6

L. Zhong, et al.
Bioorganic Chemistry 104 (2020) 104248
Fig. 5. Scanning electron microscopy images of C. albicans biofilms treated with different concentrations of c2. The inset in the 500×, 2000× panels show the area
that was magnified.
2
5
1
H, H-1), 1.68 (s, 3H, H-9), 1.65 (m, 2H, H-4), 1.61 (m, 1H, H-5), 1.52
Gingertriol B (g2): White power. 37 mg. [ ]
D
+ 16.2 (c 0.10,
1
3
1
(
(
(
m, 1H, H-2), 1.30 (s, 3H, H-10), 1.12 (m, 1H, H-2). C NMR
MeOH). H NMR (500 MHz, CDCl
3
) δ ppm 13.36 (s, 1H, 1′-OH), 13.20
125 MHz, CDCl ) δ ppm 192.1 (C-8′), 191.8 (C-9′), 169.6 (C-3′), 168.3
3
(s, 1H, 5′-OH), 10.16 (s, 1H, H-9′), 9.95 (s, 1H, H-8′), 2.71 (m, 1H, H-2),
2.69 (m, 1H, H-7′), 2.45 (dd, J = 15.5, 6.1 Hz, 1H, H-7′), 2.26 (t,
J = 5.6, 1H, H-6), 2.15 (br s, 1H, H-3), 2.13 (m, 1H, H-5), 1.90 (br dd,
J = 9.4, 4.0 Hz, 1H, H-4), 1.48 (s, 3H, H-7), 1.33 (m, 1H, H-3), 1.31 (s,
C-1′), 162.8 (C-5′), 149.3 (C-7), 109.0 (C-8), 104.3 (C-6′), 104.1 (C-4′),
9
2
8.9 (C-2′), 78.7 (C-6), 44.4 (C-3), 38.3 (C-5), 35.6 (C-1), 33.5 (C-2),
6.8 (C-4), 25.4 (C-10), 22.1 (C-7′), 21.0 (C-9). HRESIMS m/z 329.1398
-
13
[
M−H] (calcd for C19
H
22
O
5, 329.1394).
3H, H-10), 1.10 (s, 3H, H-9), 0.79 (d, J = 10.6, 1H, H-5). C NMR
2
5
Cedartriol E (c5): White power. 13 mg.[ ] + 14.5 (c 0.10, MeOH).
(125 MHz, CDCl ) δ ppm 192.1 (C-8′), 191.8 (C-9′), 168.9 (C-3′), 168.4
3
D
1
H NMR (500 MHz, CDCl
3
) δ ppm 13.44 (s, 1H, 1′-OH), 13.25 (s, 1H, 5′-
(C-1′), 166.3 (C-5′), 104.0 (C-6′), 103.9 (C-4′), 100.6 (C-2′), 88.5 (C-1),
54.9 (C-6), 40.7 (C-4), 40.5 (C-8), 34.2 (C-3), 31.9 (C-2), 28.9 (C-7),
OH), 10.16 (s, 1H, H-9′), 10.03 (s, 1H, H-8′), 2.67 (dt, J = 16.6, 6.5 Hz,
H, H-7′), 2.53 (dt, J = 16.6, 6.3 Hz, 1H, H-7′), 1.98 (m, 1H, H-4), 1.91
m, 1H, H-10), 1.87 (m, 1H, H-10), 1.81 (m, 1H, H-5), 1.67 (m, 1H, H-
), 1.38 (m, 1H, H-7), 1.33 (m, 1H, H-5), 1.33 (m, 1H, H-1), 0.97 (d,
J = 7.0, 1H, H-9), 0.96 (d, J = 7.0, 1H, H-8), 0.54 (m, 1H, H-2), 0.39
1
28.3 (C-10), 28.0 (C-5), 22.9 (C-9), 19.7 (C-7′). HRESIMS m/z 329.1397
-
(
[M−H] (calcd for C19
22
H O5, 329.1394).
2
5
4
Gingertriol C (g3): White power. 19 mg. [ ]
D
+ 21.9 (c 0.08,
1
MeOH). H NMR (500 MHz, CDCl
3
) δ ppm 13.43 (s, 1H, 1′-OH), 13.23
1
3
(
m, 1H, H-2). C NMR (125 MHz, CDCl
3
) δ ppm 192.2 (C-8′), 191.8 (C-
(s, 1H, 5′-OH), 10.15 (s, 1H, H-9′), 10.03 (s, 1H, H-8′), 2.56 (t, J = 6.7,
1H, H-7′), 2.26 (m, 1H, H-2), 2.16 (m, 1H, H-4), 2.03 (m, 1H, H-7), 2.01
(m, 1H, H-6), 1.98 (m, 1H, H-5), 1.97 (m, 1H, H-3), 1.92 (m, 1H, H-3),
9
1
2
′), 169.1 (C-3′), 168.4 (C-2′), 163.6 (C-5′), 104.5 (C-6′), 103.9 (C-4′),
01.1 (C-2′), 91.2 (C-6), 34.9 (C-3), 33.3 (C-5), 32.5 (C-7), 30.3 (C-1),
7.6 (C-10), 25.3 (C-4), 20.2 (C-9), 19.8 (C-8), 16.5 (C-7′), 13.4 (C-2).
1.86 (m, 1H, H-7), 1.60 (m, 1H, H-2), 1.30 (s, 3H, H-10), 1.26 (m, 1H,
-
13
HRESIMS m/z 329.1398 [M−H] (calcd for C19
H
22
D
O
5, 329.1394).
H-5), 1.02 (s, 3H, H-9), 0.88 (m, 1H, H-7′). C NMR (125 MHz, CDCl
3
)
2
5
Gingertriol A (g1): White power. 14 mg. [ ] + 29.2 (c 0.08,
δ ppm 192.2 (C-8′), 191.8 (C-9′), 168.9 (C-3′), 168.3 (C-1′), 163.8 (C-
5′), 104.6 (C-6′), 103.7 (C-4′), 100.8 (C-2′), 85.7 (C-1), 49.8 (C-4), 40.7
(C-6), 39.4 (C-8), 31.8 (C-7), 28.7 (C-5), 27.6 (C-10), 26.7 (C-2), 24.8
1
MeOH). H NMR (500 MHz, CDCl
3
) δ ppm 13.47 (s, 1H, 1′-OH), 13.25
(
s, 1H, 5′-OH), 10.14 (s, 1H, H-9′), 10.09 (s, 1H, H-8′), 2.63 (br d,
-
J = 16.9, 1H, H-7′), 2.41 (dd, J = 16.6, 6.5 Hz, 1H, H-7′), 1.92 (m, 1H,
H-5), 1.71 (dd, J = 12.6, 7.5 Hz, 1H, H-4), 1.45 (m, 1H, H-1), 1.43 (m,
(C-3), 23.4 (C-9), 14.9 (C-7′). HRESIMS m/z 329.1393 [M−H] (calcd
for C19
22
H O5, 329.1394).
1
8
H, H-4), 1.39 (s, 3H, H-10), 1.33 (m, 1H, H-7), 0.87 (d, J = 6.9, 3H, H-
13
), 0.83 (d, J = 6.9, 3H, H-9), 0.58 (m, 1H, H-2), 0.57 (m, 1H, H-2).
) δ ppm 192.1 (C-8′), 191.6 (C-9′), 169.5 (C-3′),
68.3 (C-1′), 162.6 (C-5′), 103.9 (C-6′), 103.8 (C-4′), 98.1 (C-2′), 89.3
C
4
.2. Agar disk diffusion test
NMR (125 MHz, CDCl
3
1
Disk diffusion test was performed as previously described [19]. In
(
C-6), 34.6 (C-5), 33.5 (C-3), 32.6 (C-1), 32.4 (C-7), 31.1 (C-4), 22.1 (C-
5
brief, 500 μL C. albicans (SC5314) suspension (5 × 10 cells/mL) was
spread uniformly onto yeast peptone dextrose (YPD) agar plates. A plate
with 1% DMSO was used as the vehicle control. The 6-mm-diameter
1
3
0), 20.1 (C-9), 19.7 (C-8), 16.2 (C-7′), 14.3 (C-2). HRESIMS m/z
-
29.1398 [M−H] (calcd for C19
22
H O5, 329.1394).
7

L. Zhong, et al.
Bioorganic Chemistry 104 (2020) 104248
filter disks contain different content crude product (200 μg, 100 μg,
4.7. Scanning electron microscopy (SEM) observations
5
2
0 μg) was placed onto the agar surface. The plates were incubated for
4 h at 35 °C and then measure the growth inhibition zones. Images
SEM was performed to investigate the ultrastructure of C. albicans
YEM12 biofilms [44–45]. Sterile glass disks coated with poly-L-lysine
hydrobromide (Sigma) were used to incubate the biofilms. Then the
medium of each well was removed, biofilms were washed with PBS and
fixed with 2.5% glutaraldehyde for 2 h. Later, biofilms were rinsed
twice in cacodylate buffer, garnished with 1% osmic acid for 2 h, de-
hydrated in an ascending ethanol series, treated with hexamethyldisi-
lazane (Polysciences Europe GmbH, Eppelheim, Germany), and dried
overnight. The specimens were coated with gold. Afterwards, the ul-
trastructure of the biofilms was visualized with a Carl Zeiss SEM (EVO
LS10) in high-vacuum mode.
were collected by using Chemidoc XRS +.
4.3. In vitro antifungal assay
The minimum inhibitory concentrations (MIC) of compounds
againest fungal were determined by the broth microdilution method
based on the Clinical and Laboratory Standards Institute (CLSI) stan-
dard M27-A3 [39]. A range of concentrations from 50 to 1.5625 μg/mL
for synthesised compounds were made in RPMI 1640. One hundred
microliter compound and 100 µL cells suspension with a final con-
3
centration of 2.5 × 10 cells/mL were added into 96-well plates. Flu-
Declaration of Competing Interest
conazole was used as positive control drugs. Then, the plates were in-
cubated at 35 °C for 24 h. Optical densities at 540 nm (OD540) were
measured by a microplate reader (Tecan SUNRISE) and the MIC50 was
defined as the concentration of drugs that inhibited 50% of cell growth.
All tests were repeated for three times. Compounds were dissolved in
DMSO. The maximum amounts of DMSO that was added into the cul-
ture medium did not affect cell multiplication.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This research work was funded by the National Natural Science
Foundation of China (81773901), the Drug Innovation Major Project
4
.4. Cytotoxicity assay
(
2018ZX09711-001-007 and 2018ZX09735002-003) and the 111
Cytotoxicity was measured using the MTT [3-(4, 5- dimethylthiazol-
-yl)-2, 5-diphenyl tetrazolium bromide] method [40]. In brief, the LO2
Project from Ministry of Education of China and the State
Administration of Foreign Export Affairs of China (B18056).
2
(
normal human hepatic cell line) cell lines were seeded into 96-well
4
plates (10 cells/well). After incubating over night, the cells were
treated with compounds and further incubated for 48 h at 37 °C. Then,
the MTT solution was added to each well. After 4 h, DMSO was added to
dissolve formazan crystals. The absorbance of wells was measured at
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104248.
5
70 nm.
References
4.5. In vitro antibiofilm assay
[
[
1] D.J. Newman, G.M. Cragg, J. Nat. Prod. 79 (2016) 629–661.
2] L.L. Silver, Future Microbiol. 10 (2015) 1711–1718.
The antibiofilm effect of c2 was measured by a crystal violet (CV)
assay [41]. In brief, 100 μL of C. albicans YEM12 cell suspension
[3] D.W. Denning, Lancet. 362 (2003) 1142–1151.
6
[4] V. Moudgal, J. Sobel, Expert Opin. Pharmacother. 11 (2010) 2037.
(
1.0 × 10 cells/mL in RPMI 1640 medium) was placed into 96-well
[
5] F. Benz, A. Knusel, J. Nuesch, H. Trelchler, W. Voser, Helvetica Chim. Acta 57
plates and incubated at 37 °C for 1.5 h for initial adhesion. The medium
was aspirated and nonadherent cells were removed by washing with
(
1974) 2459–2477.
[6] T. Iwamoto, A. Fujie, K. Sakamoto, Y. Tsurumi, N. Shigematsu, M. Yamashita,
S. Hashimoto, M. Okuhara, M. Kohsaka, J. Antibiot. 47 (1994) 1084–1091.
1
00 μL of PBS. 100 μL RPMI 1640 medium with or without different
[
[
7] E.M. Carmona, A.H. Limper, Clin. Chest Med. 38 (2017) 393–402.
8] D. Allen, D. Wilson, R. Drew, J. Perfect, Expert Rev. Anti-infect. Ther. 13 (2015)
787–798.
concentrations of c2 was added to each well and further incubated for
4 h. For detection of c2 on mature biofilms, biofilms pregrown for 24 h
were treated with different concentrations of c2 and were incubated for
2
[9] N.M. Revie, K.R. Iyer, N. Robbins, L.E. Cowen, Curr. Opin. Microbiol. 45 (2018)
7
0–76.
10] D. Marchaim, L. Lemanek, S. Bheemreddy, K.S. Kaye, J.D. Sobel, Obstet. Gynecol.
20 (2012) 1407–1414.
[11] N. Liu, J. Tu, G.Q. Dong, Y. Wang, C.Q. Sheng, J. Med. Chem. 61 (2018)
484–5511.
12] S. Yin, J.J. Xue, C.Q. Fan, Z.H. Miao, J. Ding, J.M. Yue, Org. Lett. 9 (2007)
549–5552.
[13] Y. Yu, L.S. Gan, S.P. Yang, L. Sheng, Q.F. Liu, S.N. Chen, J. Li, J.M. Yue, J. Nat. Prod.
9 (2016) 1365–1372.
14] I.P. Singh, J. Sidana, S.B. Bharate, W.J. Fole, Nat. Product Reports 27 (2010)
93–416.
[15] Z.C. Shang, M.H. Yang, K.L. Jian, X.B. Wang, L.Y. Kong, Chem. Eur. J. 22 (2016)
2
4 h at 37 °C. Then the content of each well was removed, wells were
[
washed twice with PBS and fixed the cell with 99% methanol (200 μL)
for 15 min. The 99% methanol was discarded, and each well was
stained with 200 μL of 0.1% crystal violet solution followed by 30 min
incubation at room temperature. Later, the excess crystal violet solution
from the plates was removed, thoroughly washed with distilled water
for 3 to 4 times and air dried at room temperature. The crystal violet
stained biofilm was solubilized in 33% acetic acid (200 μL). Afterwards,
1
5
[
5
7
[
3
1
00 μL of the destaining solution was transferred to a new 96-well
1
1778–11784.
plate, and the absorbance was recorded at 570 nm. The experiments
were carried out in triplicates.
[
[
16] Z.C. Shang, M.H. Yang, R.H. Liu, X.B. Wang, L.Y. Kong, Sci. Rep. 6 (2016) 39815.
17] Z.C. Shang, C. Han, J.L. Xu, R.H. Liu, Y. Yin, X.B. Wang, M.H. Yang, L.Y. Kong,
Phytochemistry. 163 (2019) 111–117.
[
[
[
18] R.H. Liu, Z.C. Shang, T.X. Li, M.H. Yang, L.Y. Kong, Antimicrob. Agents Chemother.
4
.6. Confocal laser scanning microscopy (CLSM) observations
6
1 (2017) 2707–2716.
19] J.L. Xu, R.H. Liu, F.J. Sun, L. An, Z.C. Shang, L.Y. Kong, M.H. Yang, Front. Cell.
Infect. Microbiol. 9 (2019) 211.
CLSM was performed by using six-well plates containing plastic
20] W.Q. Chang, J. Liu, M. Zhang, H.Z. Shi, S. Zheng, S.Y. Jin, Y.H. Gao, S. Wang, A. Ji,
H.X. Lou, Nat. Commun. 9 (2018) 5102.
disks as previously reported [42–43]. Biofilms (C. albicans YEM12)
were incubated in plates following the method described above. After
incubation each well was washed with PBS, stained with 10 μg/mL
fluorescein diacetate (FDA) and 5 μg/mL propidium iodide (PI) for
[21] B.X. Liang, W.Y. Shao, C.G. Zhu, G.S. Wen, X. Yue, R.M. Wang, J.M. Quan, J. Du,
X.Z. Bu, ACS Chem. Biol. 11 (2016) 425–434.
[
22] L. Leanza, M. Romio, K.A. Becker, M. Azzolini, L. Trentin, A. Manago, E. Venturini,
A. Zaccagnino, A. Mattarei, L. Carraretto, A. Urbani, S. Kadow, L. Biasutto,
V. Martini, F. Severin, R. Peruzzo, V. Trimarco, J.H. Egberts, C. Hauser, A. Visentin,
G. Semenzato, H. Kalthoff, M. Zoratti, E. Gulbins, C. Paradisi, I. Szabo, Cancer Cell.
3
0 min and viewed under confocal laser scanning microscope (LSM800;
Carl Zeiss).
8

L. Zhong, et al.
Bioorganic Chemistry 104 (2020) 104248
3
1 (2017) 516–531.
[34] Q. Cheng, J.K. Snyder, J. Org. Chem. 53 (1988) 4562–4567.
[
[
23] M. Fridman, R.I. Benhamou, M. Bibi, J. Berman, Angew. Chem. Int. Ed. 57 (2018)
[35] R. Cai, M.M. Hu, Y.J. Zhang, C. Niu, T.L. Yue, Y.H. Yuan, Z.L. Wang, LWT-Food Sci.
Technol. 106 (2019) 50–56.
6
230–6235.
24] A.L. Lawrence, R.M. Adlington, J.E. Baldwin, V. Lee, J.A. Kershaw, A.L. Thompson,
Org. Lett. 12 (2010) 1676–1679.
[36] A. Thakre, G. Zore, S. Kodgire, R. Kazi, S. Mulange, R. Patil, A. Shelar,
B. Santhakumari, M. Kulkarni, K. Kharat, S.M. Karuppayil, Med Mycol. 56 (2018)
565–578.
[
[
[
[
25] S.L. Schreiber, Science. 287 (2000) 1964–1969.
26] M.D. Burke, S.L. Schreiber, Angew. Chem. Int. Ed. 43 (2004) 46–58.
27] W.R.J. Galloway, I.L. Albert, R.S. David, Nat. Commun. 1 (2010) 80.
28] Y.L. Zhang, C. Chen, X.B. Wang, L. Wu, M.H. Yang, J. Luo, C. Zhang, H.B. Sun,
J.G. Luo, L.Y. Kong, Org. Lett. 18 (2016) 4068.
[37] M. Gulati, C.J. Nobile, Microbes Infect. 18 (2016) 310–321.
[38] Y. Lu, C. Su, H.P. Liu, Trends Microbiol. 22 (2014) 707–714.
[39] Clinical and Laboratory Standards Institute, Reference method for broth dilution
antifungal susceptibility testing of yeasts, approved standard 3rd ed., Document
M27-A3, Clinical and Laboratory Standards Institute, Wayne, PA, 2008.
[40] T. Mosmann, J. Immunol. Methods 65 (1983) 55–63.
[41] A. Bruder-Nascimento, C.H. Camargo, A.L. Mondelli, M.F. Sugizaki, T. Sadatsune,
E. Bagagli, Braz. J. Microbiol. 45 (2014) 1371–1377.
[
[
29] L. Wu, J. Luo, X.B. Wang, R.J. Li, Y.L. Zhang, L.Y. Kong, RSC. Adv. 5 (2015)
9
3900–93906.
30] L. Wu, J. Luo, Y.L. Zhang, M.D. Zhu, X.B. Wang, J.G. Luo, M.H. Yang, B.Y. Yu,
H.Q. Yao, Y. Dai, Q.L. Guo, Y.J. Chen, H.B. Sun, L.Y. Kong, Tetrahedron Lett. 56
(
2015) 229–232.
[42] D.N. Jackson, L. Yang, S.B. Wu, E.J. Kennelly, P.N. Lipke, Antimicrob. Agents.
Chemother. 59 (2015) 6032–6038.
[
[
[
31] J. Novak, C. Bitsch, J. Langbehn, F. Pank, M. Skoula, Y. Gotsiou, C. Frantz, Biochem.
Synst. Ecol. 28 (2000) 697–704.
[43] N.N. Mishra, S. Ali, P.K. Shukla, Braz. J. Infect. Dis. 18 (2014) 287–293.
[44] K.L. Lee, H.R. Buckley, C.C. Campbell, Sabouraudia. 13 (1975) 148–153.
[45] L.X. Zhao, D.D. Li, D.D. Hu, G.H. Hu, L. Yan, Y. Wang, Y.Y. Jiang, PLoS. One. 8
(2013) 79671.
32] M. Kozuka, T. Sawada, E. Mizuta, F. Kasahara, T. Amano, T. Komiya, M. Goto,
Chem. Pharm. Bull. 30 (1982) 1964–1973.
33] R.S. Xu, J.K. Snyder, K. Nakanishi, J. Am. Chem. Soc. 106 (1984) 734.
9