Design, synthesis and biological evaluation of 2-substituted 3-hydroxy-6-methyl-4H-pyran-4-one derivatives as Pseudomonas aeruginosa biofilm inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.09.041

Drug-resistant bacteria associated with biofilm formation are rapidly on the rise, requiring novel therapeutic options to combat biofilm induced drug-resistance. In this study, a class of 3-hydroxy-2-(phenylhydroxy-methyl)-6-methyl-4H-pyran-4-one derivati

Full text of DOI:10.1016/j.ejmech.2018.09.041

Accepted Manuscript
Design, synthesis and biological evaluation of 2-substituted 3-hydroxy-6-methyl-4H-
pyran-4-one derivatives as Pseudomonas aeruginosa biofilm inhibitors
Yi-Bin Li, Jun Liu, Zhi-Xing Huang, Jia-Hui Yu, Xiao-Fang Xu, Ping-Hua Sun, Jing Lin,
Wei-Min Chen
PII:
S0223-5234(18)30813-4
10.1016/j.ejmech.2018.09.041
EJMECH 10752
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 May 2018
Revised Date: 24 July 2018
Accepted Date: 14 September 2018
Please cite this article as: Y.-B. Li, J. Liu, Z.-X. Huang, J.-H. Yu, X.-F. Xu, P.-H. Sun, J. Lin, W.-M.
Chen, Design, synthesis and biological evaluation of 2-substituted 3-hydroxy-6-methyl-4H-pyran-4-one
derivatives as Pseudomonas aeruginosa biofilm inhibitors, European Journal of Medicinal Chemistry
(2018), doi: https://doi.org/10.1016/j.ejmech.2018.09.041.
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Graphical Abstract
Design, synthesis and biological evaluation of 2-substituted
3-hydroxy-6-methyl-4H-pyran-4-one derivatives as Pseudomonas
aeruginosa biofilm inhibitors
Yi-Bin Li1, Jun Liu1, Zhi-Xing Huang, Jia-Hui Yu, Xiao-Fang Xu, Ping-Hua Sun, Jing Lin*,
Wei-Min Chen*
College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China
*Corresponding authors. Tel.: +86 20 8522 1367(J. Lin), +86 20 8522 4497 (W.-M. Chen).
Fax: +86 20 8522 4766.
E-mail address: linjing_jnu@163.com (J. Lin), twmchen@jnu.edu.cn(W.-M. Chen).

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Design, synthesis and biological evaluation of 2-substituted
3-hydroxy-6-methyl-4H-pyran-4-one derivatives as Pseudomonas
aeruginosa biofilm inhibitors
Yi-Bin Li1, Jun Liu1, Zhi-Xing Huang, Jia-Hui Yu, Xiao-Fang Xu, Ping-Hua Sun, Jing Lin*,
Wei-Min Chen*
College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China
1 These authors contributed equally to this work.
*Corresponding authors. Tel.: +86 20 8522 1367(J. Lin), +86 20 8522 4497 (W.-M. Chen).
Fax: +86 20 8522 4766.
E-mail address: linjing_jnu@163.com (J. Lin), twmchen@jnu.edu.cn (W.-M. Chen).
Abstract
Drug-resistant bacteria associated with biofilm formation are rapidly on the rise,
requiring novel therapeutic options to combat biofilm induced drug-resistance. In this
study, a class of 3-hydroxy-2-(phenylhydroxy-methyl)-6-methyl-4H-pyran-4-one
derivatives (1a-1e) were found by screening of an in-house compound library to be
potential Pseudomonas aeruginosa biofilm inhibitors. Thirty one novel 2-substituted
3-hydroxy-6-methyl-4H-pyran-4-one derivatives were synthesized and assayed for
their biofilm inhibitory activity. A promising biofilm inhibitor 6a was identified, and
showed an obvious biofilm inhibitory effect even at a concentration of 2.5 µM.
Further mechanism studies revealed that 6a only shows inhibitory effects on the
expression of pqsA-gfp in a fluorescent reporter strain, and the production of a PQS-
regulated virulence factor, pyocyanin. This indicates that this type of compound
exercises its anti-biofilm activity specifically through the PQS pathway. Novel
chemical biofilm inhibitors are described here and guard against biofilm formation
associated with Pseudomonas aeruginosa infections.
Keywords: Pseudomonas aeruginosa; quorum sensing; PQS pathway; biofilm
inhibitor; synthesis

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1. Introduction
Bacterial resistance infection in humans, especially the chronic infection which
show increased tolerance toward antibiotic treatments and high mortality [1] has
become a major challenge in drug discovery. Pseudomonas aeruginosa is a
powerful gram-negative opportunistic bacterium that remarkably infects
immune-compromised patients suffering from cystic fibrosis (CF) [2], burn wounds [3]
or HIV [4] and induces chronic infections. During such chronic infections, P.
aeruginosa bacteria often behaves in a biofilm manner to embed themselves in a
matrix composed mainly of exopolysaccharides, extracellular DNA and polypeptides.
This growth pattern has a protective effect and helps bacteria escape the host immune
response and thus resist antimicrobial treatment [5]. Consequently, inhibition of
bacterial biofilm formation by targeting nonmicrobicidal mechanisms may potentially
be a means of overcoming bacterial resistance.
For P. aeruginosa, the central processes in biofilm development are regulated by
the action of the quorum sensing (QS) system. P. aeruginosa possesses three main QS
component systems, las, rhl and PQS which are mediated by three chemically distinct
classes of signal molecules, N-(3-oxododecanoyl)-L-homoserine lactone
(3-oxo-C12-HSL),
N-butanoyl-L-homoserine
lactone
(C4-HSL)
and
2-heptyl-3-hydroxy-4(1H) quinolone (PQS), respectively [4, 6-11]. The third signal
molecule, PQS is unique to P. aeruginosa, and commonly referred as the
pseudomonas quinolone signal. PQS can interact with the two acyl homoserine
lactones (AHLs) systems (las and rhl) and provide a regulatory link between these
two AHL-based QS systems [12]. PQS can also act as an iron chelator, affecting iron
transport and regulating biofilm formation. It is known that an increase in readily
bioavailable iron can promote the formation of biofilm [13]. PQS is therefore likely to
be a key element with multiple functions in the regulation of biofilm formation in P.
aeruginosa. In addition, it has been demonstrated that PQS-deficient mutant P.
aeruginosa forms less biofilm, and PQS is produced in high amounts in the sputum of
patients with cystic fibrosis [14]. These facts provide a rationale for efforts to discover
drugs that target PQS regulation.
The biosynthesis of PQS in P. aeruginosa requires proteins encoded by the genes
of pqsABCDE which are under the control of the transcriptional activator PqsR [12,
15-18]. When the concentration of PQS reaches a certain level, the transcriptional
regulator PqsR is activated and induces the production of virulence factors like

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pyocyanin [19] and biofilm [20] and drives forward the expression of the pqsA, the
first gene in the PQS biosynthetic operon [21]. Therefore, PQS system regulators,
especially the PqsR-mediated negative transcriptional regulator, would be promising
agents with which to control P. aeruginosa persistent infections.
In this study, based on the novel hydroxypyrone biofilm inhibitors that have been
identified by screening, four series of new 2-substituted 3-hydroxy-6-methyl-
4H-pyran-4-one derivatives were designed and synthesized, and a promising biofilm
inhibitor (6a) was identified. This compound shows excellent biofilm inhibitory
behavior and was found in a bioreporter assay to inhibit pqsA expression, based on the
expression of a green fluorescent protein (GFP) in P. aeruginosa (pqsA-gfp), and the
production of the PQS-regulated virulence factor pyocyanin.
2. Chemistry
Four series of 2-substituted 3-hydroxy-6-methyl-4H-pyran-4-one derivatives,
based on the novel hydroxypyrone biofilm inhibitors that were identified through
screening have been designed and synthesized (Fig 1.). The hydroxypyrone ring is
similar to the quinoline ring in 2-heptyl-3-hydroxy-4(1H) quinolone (PQS), which is
the core structure of the signal molecules, and we speculated that the hydroxypyrone
moiety in the designed derivatives is possibly the pharmacophore in biofilm inhibitors.
Accordingly, we selected the hydroxypyrone skeleton as a starting point for further
optimization with the following aims: (1) to investigate the effect of the substituent
positions at the 2-substituted position on the benzene ring, compounds in series I
(5a-5d) were designed; (2) to introduce different groups on the phenyl ring obtaining
compounds in series II (6a-6q) to investigate the effect of substituent types; (3) to
design compounds in which the hydrophobic phenyl ring is replaced by a fatty acid
chain, because PQS signals molecules with such a chain, compounds in series III
(7a-7f) were synthesized; (4) to examine the role of the α-hydroxyl group, compounds
in series IV (8a-8d) lacking the α-hydroxyl group were designed. (Fig 2).
[Figure 1]
[Figure 2]
2.1. Synthesis of the target compounds in series I-III
The synthesis of the precursor allomaltol (2) was performed in two steps, starting

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with the reaction of kojic acid (5-hydroxy-2-hydroxylmethyl-4H-pyran-4-one) with
thionyl chloride followed by reduction with zinc under acidic conditions [22]. Due to
the higher reactivity of C-6 on the pyrone ring of 2 when compared to C-3,
appropriate functionalization can easily be achieved with an aldol reaction [23].
Synthesis of compounds 5a-5d, 6a-6p, 7a-7f were achieved by condensation of
allomaltol (2) (1.0 eq) with different aldehydes (1.2 eq) in the presence of a catalyst,
1,4-diazabicyclo[2.2.2]octane (DABCO, 1.2 eq).
[Scheme 1]
2.1. Synthesis of target compounds in series IV
Synthesis of compounds 8a-8e were achieved by condensation of allomaltol (2)
(1.0 eq) with different aldehydes (1.2 eq) in the presence of a catalyst,
1,4-diazabicyclo[2.2.2]octane (DABCO, 1.2 eq), and subsequently, the α–hydroxyl
group was reduced using triethylsilane (5.0 eq.) and trifluoroacetic acid (5.0 eq.) in
CH2Cl2.
[Scheme 2]
3. Result and Discussion
3.1. Anti-Biofilm Screening and Structural-Activity Relationship (SAR) study
As shown in Table 1, derivatives with substituents at the para-position of the
benzene ring presented good biofilm inhibitory effects. Among these, compound with
a nitro group on the benzene ring (6a) displayed the best anti-biofilm effect, with an
inhibition rate of 50.98 ± 1.97%, and derivatives with the hydrophobic aromatic ring
substituted on the benzene ring show relatively low activity with the exception of 6d,
whose aromatic substituent is a nitrophenyl ring. Derivatives 5a-5d with substituents
on the meta- or ortho- position of the benzene ring showed little or no biofilm
inhibitory effect.
Next, to further explore the flexibility of the chain in R1 on the anti-biofilm
activity, compounds 6f-6k were synthesized. The anti-biofilm results showed that 6j
and 6k were more active than 6f, 6g, 6h and 6i, which indicates that rigidity in this
side chain are beneficial to the activity. For compounds with a rigid acetylene linkage

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in this side chain, various substituents on the phenyl ring, including those with
electron-withdrawing groups (6l, 6m, 6n), or with electronic donating groups (6o) and
a substituted site (6m vs. 6l) all show only a slight impact on the anti-biofilm activity.
When the phenyl ring was replaced by a cyclohexane (6p) or pyridine ring (6q), the
anti-biofilm activity was enhanced.
For series III (7a-7f) of the compounds with 3-7 carbons in the alkyl chain,
derivatives with chain lengths of 5-6 carbons have relatively good anti-biofilm effects.
The inhibition rate value of compounds 7c and 7d were 44.65 ± 2.48 % and 41.45% ±
1.77 %, respectively. When compounds 1a-1d were compared with compounds 8a-8d,
it was found that reduction of the chiral α-hydroxyl has no effect on activity.
Based on these results, some preliminary structural-activity relationships (SAR)
of the 2-substituted 3-hydroxy-6-methyl-4H-pyran-4-one derivatives could be derived
and are summarized thus: (1) only the para-substituent on the benzene ring is
conducive to activity; (2) a nitro group promotes the activity significantly, and
aromatic R groups are generally active; (3) when the hydrophobic group is a alkyl
chain, as in series III, a chain length of 5-6 carbons is associated with better activity;
and (4) the chiral α-hydroxyl group is not essential for activity.
Finally, we tested the effect of compounds on bacterial growth and found that all
derivatives, even at a concentration of 512 µM have no effect on the growth of P.
aeruginosa (PAO1). These results illustrated that 2-substituted 3-hydroxy-6-methyl-
4H-pyran-4-one derivatives inhibit bacterial biofilm formation through non-
microbicidal mechanisms.
[Table 1]
3.2. Effect of 6a on biofilm formation and growth.
To confirm the anti-biofilm activity of these novel 2-substituted
3-hydroxy-6-methyl-4H-pyran-4-one derivatives, the most active compound (6a) was
selected for further experiments with concentration gradients. We first tested the
inhibitory effects of 6a on the biofilm formation of P. aeruginosa PAO1 at 40, 20, 10,
5, 2.5 and 0 µM after treatment for 24 h. We found that 6a significantly reduces
biofilm formation in a dose-dependent manner. It exerts excellent biofilm inhibitory
activity, and at concentrations above 20 µM, the inhibition exceeds 50%, and a
relatively low but still obvious biofilm inhibitory activity was observed at 2.5 µM, a

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concentration at which the inhibition rate is still over 30% (Fig 3A). This effect of 6a
on the formation of P. aeruginosa biofilm was observed using Confocal Laser
Scanning Microscope (CLSM) images. As shown in Fig 3C, after the treatment with
either the maximum concentration (40 µM) or the minimum concentration (2.5 µM)
of 6a, the number of bacteria that aggregated to form a biofilm were reduced in a
dose-dependent manner, which further demonstrated the good anti-biofilm effect of
this class of compounds. In addition, the effect of 6a on P. aeruginosa PAO1 growth
was evaluated by monitoring the OD of cultures at 600 nm. The results showed that
treatment with the 40, 20, 10, 5 or 2.5 µM of 6a had no inhibitory effect on bacterial
growth (Fig 3B).
[Figure 3]
3.3 Effect of 6a on QS system report strains
Since 6a displayed the best anti-biofilm activity among the synthesized
compounds, it was also used as a model in the anti-biofilm mechanism study. As
mentioned above, QS systems including las, rhl, and PQS pathways are the key
systems that are responsible for the regulation of biofilm formation and virulence
factors secretion in P. aeruginosa. It is known that the lasB gene, which encodes
elastase is mainly controlled by the transcriptional activator LasR while rhlA and
pqsA are the first genes of respectively, the rhl operon and pqs operon that code for
the production of rhamnolipid and the PQS molecule. Thus to explore the biofilm
inhibitory mechanism of these compounds, the regulated effects of 6a on the three QS
systems were evaluated using three reporter strains PAO1-lasB-gfp, PAO1-rhlA-gfp
and PAO1-pqsA-gfp [24], in which the green fluorescence protein (gfp) gene was
integrated downstream of the promoter of lasB, rhlA and pqsA respectively, and the
production of GFP protein can reflect the activity of these promoters. As shown in Fig.
4A-4C, compound 6a significantly inhibits the fluorescence of PAO1-pqsA-gfp strain
in a dose-dependent manner, while the expression of lasB-gfp and rhlA-gfp were not
obviously affected by 6a in the same experiment. To further verify that 6a indeed
targets the corresponding QS genes but not the GFP protein itself, PAO1-gfp strain
was used as a control strain to investigate the influence of 6a on the fluorescence of
GFP protein. The result showed that 6a has no effect on the fluorescence of GFP (Fig.
4D). Taken together, our bioreporter assays indicate that this novel 2-substituted

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3-hydroxy-6-methyl-4H-pyran-4-one derivative possesses the anti-biofilm activity
expressed specifically through the PQS pathway.
[Figure 4]
3.4 Effect of 6a on P. aeruginosa virulence
To further verify the anti-biofilm mechanism of 6a specific to the PQS pathway,
we measured the production of three virulence factors (elastase, rhamnolipid and
pyocyanin), which is directly controlled by the las, rhl and PQS system respectively.
Consistent with the reported strain assay results, the production of pyocyanin that is
strictly under the control of pqs pathway, was significantly reduced in a dose-
dependent manner by treatment with sub-micromolar concentrations (0, 2.5, 5, 10, 20
and 40 µM) of 6a. At 40 µM, treatment with 6a leads to a 60% decrease in pyocyanin
production compared with the control, while 6a has almost no effect on the
production of elastase and rhamnolipid, which are under the control of las and rhl
system, respectively (Fig. 5A-5C). The exclusively inhibitory effect of 6a on the
production of pyocyanin further demonstrated that 6a exerted the anti-biofilm effect
through the pqs pathway.
[Figure 5]
3.5 Cytotoxicity of 6a
Finally we evaluated the toxicity of 6a in mammalian cells using murine
macro-phage RAW264.7 cell and an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay. It can be seen from Fig. 6 that no cytotoxic effect
was observed at concentration of 2.5 µM or lower, and only weak cytotoxicity appears
at 50 µM. This results illustrate that 6a is not cytotoxic at concentrations where
biofilm inhibitory activity is observed.
[Figure 6]
4. Conclusion
Clinically, and despite prevention many biofilm-associated infections occur
under a variety of circumstances, but once a biofilm has been established on an abiotic
or tissue surface, eradication is nearly impossible with conventional doses of

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antibiotics, and this condition can eventually lead to death [25]. Combinatorial
treatment with antibiotics and compounds exhibiting anti-biofilm activity is
considered as a potential strategy to solve this problem. In this study, novel
2-substituted 3-hydroxy-6-methyl-4H-pyran-4-one derivatives were identified as
potential anti-biofilm agents. A study of the structure-activity relationships (SAR)
revealed that compounds with the rigid long hydrophobic substituent, especially with
a nitro group, at the para position of the phenyl ring, is the most favorable for
anti-biofilm activity. Using 6a as a model molecule which was shown to have efficient
P. aeruginosa biofilm inhibitory activity but no growth inhibitory effect, the PQS
pathway specific anti-biofilm mechanism of this type of compound was revealed. It
was shown that 6a exclusively reduces the production of the extracellular virulence
factor pyocyanin and the expression of pqsA-gfp. In summary, this study has
identified a new class of biofilm inhibitors, which provides a new means to reduce the
pathogenicity of P. aeruginosa.
5. Experimental section
5.1. Chemical Synthesis
All chemicals were purchased from Alfa Aesar or Sigma Aldrich. The synthetic
routes to 2-substituted 3-hydroxy-6-methyl-4H-pyran-4-one derivatives are shown in
1
Scheme 1 and Scheme 2. All the compounds were identified through H NMR and
13C NMR spectroscopy and high resolution mass spectrometry (ESI-HRMS) before
biological testing. Unless otherwise specified, proton (1H) and carbon (13C) NMR
o
spectra were recorded at 18 C in base-filtered DMSO-d6 with a Varian spectrometer
1
13
operating at 300 MHz for H NMR and 75 MHz for C NMR. Low resolution ESI
mass spectra were recorded on a Finnigan LCQ Advantage MAX mass spectrometer
fitted with a 4000 Q TRAP or Agilent 6130 Quadruple LC/MS. High resolution mass
spectra were obtained on an Agilent 6210 series LC/MSD TOF mass spectrometer.
Melting points were determined with a digital melting point apparatus (Briwell/GH60)
and are uncorrected. A high performance liquid chromatograph (HPLC) apparatus was
used to characterize their purity. The purity of all compounds used in biological assays
was confirmed to be ≥95%.

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5.1.1. General procedure for the reaction of 1a-1e, 5a-5d, 6a-6q, 7a-7f (procedure A)
A mixture of allomaltol (5-hydroxy-2-methyl-4H-pyran-4-one, 4) (2.00 mmol)
and the relevant aldehyde (2.40 mmol) was stirred in the presence of DABCO (2.40
mmol) in dioxane:H2O (1:1, 10 mL) at rt for 12 h. The product was extracted with
EtOAc (3 × 20 mL) after the reaction was complete. The combined organic layer was
dried with anhydrous Na2SO4, concentrated in vacuo and purified by column
chromatography (MeOH:DCM=0.5:9.5) to afford a solid.
5.1.1.1. 3-Hydroxy-2-(hydroxy(phenyl)methyl)-6-methyl-4H-pyran-4-one (1a)
The reaction was performed according to the general procedure A, using 4 (1.00 g,
7.90 mmol) and benzaldehyde (0.97 mL, 9.48 mmol). The crude product was purified
by column chromatography (MeOH:DCM=0.5:9.5) to afford a white solid 1a. (1.56 g,
85.6%): mp 170.3-171.1 oC; 1H NMR (300 MHz, DMSO-d6)
δ
2.18 (s, 3H, CH3), 6.00
13
(s, 1H, CH-OH), 6.20 (s, 1H, C=CH), 7.27-7.43 (m, 5H, Ph-H); C NMR (75 MHz,
DMSO-d6)
δ 174.3 (C=O), 165.0 (CH3-C=CH), 151.0 (C-OH), 141.8 (Ph-C1’),
141.0 (HOCHC=COH), 128.7 (Ph-C3’), 127.8 (Ph-C4’), 126.4 (Ph-C2’), 111.6
(CH3=CH), 66.3 (CH-OH), 19.7 (CH3); ESI-MS m/z: 233.0 [M+H]+; ESI-HRMS m/z:
233.0804 [M+H]+, calcd for C13H13O4 233.0808.
5.1.1.2.
2-((4-Fluorophenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-one
(1b)
The reaction was performed according to the general procedure A, using 4 (1.00 g,
7.90 mmol) and p-fluorobenzaldehyde (1.00 mL, 9.48 mmol). The crude product was
purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a white solid 1b.
1
(1.79 g, 90.2%): mp 151.4-152.2 oC; H NMR (300 MHz, DMSO-d6)
δ 2.19 (s, 3H,
CH3), 6.00 (s, 1H, CH-OH), 6.20 (s, 1H, C=CH), 7.14-7.20 (t, J = 9.0 Hz, 2H, Ph-H),
13
7.41-7.46 (dd, J = 8.4 Hz, J = 5.4 Hz, 2H, Ph-H); C NMR (75 MHz, DMSO-d6)
δ
174.3 (C=O), 165.0 (CH3-C=CH), 160.3 (Ar-C4’), 150.7 (C-OH), 141.1
(HOCHC=COH), 138.0 (Ar-C1’), 128.4 (Ar-C2’), 115.6 (Ar-C3’), 115.3 (Ar-C3’),
111.6 (CH3=CH), 65.7 (CH-OH), 19.7 (CH3); ESI-MS m/z: 251.0 [M+H]+;
ESI-HRMS m/z: 251.0710 [M+H]+, calcd for C13H13FO4 251.0714.
5.1.1.3.
2-((4-Chlorophenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-one
(1c)
The reaction was performed according to the general procedure A, using 4 (1.00 g,
7.90 mmol) and 4-chlorobenzaldehyde (1.21 g, 9.48 mmol). The crude product was

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purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a white solid 1c.
(1.85 g, 87.5%): mp 152.5-153.0 oC; 1H NMR (300 MHz, DMSO-d6)
δ
2.18 (s, 3H,
13
CH3), 6.00 (s, 1H, CH-OH), 6.20 (s, 1H, C=CH), 7.41 (s, 4H, Ph-H); C NMR (75
MHz, DMSO-d6)
δ 174.3 (C=O), 165.0 (CH3-C=CH), 150.5 (C-OH), 141.2
(HOCHC=COH), 140.8 (Ar-C1’), 132.3 (Ar-C4’), 128.7 (Ar-C3’/C5’), 128.2
(Ar-C2’/C6’), 111.6 (CH3=CH), 65.7 (CH-OH), 19.7 (CH3); ESI-MS m/z: 267.0
[M+H]+; ESI-HRMS m/z: 267.0418 [M+H]+, calcd for C13H12ClO4 267.0419.
5.1.1.4.
3-Hydroxy-2-(hydroxy(4-(trifluoromethyl)phenyl)methyl)-6-methyl-4H-pyran-4-one
(1d)
The reaction was performed according to the general procedure A, using 4 (1.00 g,
7.90 mmol) and 4-(trifluoromethyl)benzaldehyde (1.30 ml, 9.48 mmol). The crude
product was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a
white solid 1d. (2.05 g, 89.1%): mp 179.7-180.6 oC; 1H NMR (300 MHz, DMSO-d6)
2.17 (s, 3H, CH3), 6.10 (s, 1H, CH-OH), 6.21(s, 1H, C=CH), 7.60-7.63 (d, J = 8.4 Hz,
2H, Ph-H), 7.71-7.74 (d, J = 8.4 Hz, 2H, Ph-H); 13C NMR (75 MHz, DMSO-d6)
δ
δ
174.3 (C=O), 165.2 (CH3-C=CH), 150.1 (C-OH), 146.5 (Ar-C1’), 141.4
(HOCHC=COH), 128.6 (Ar-C4’), 127.1 (Ar-C3’/C5’), 126.5 (Ar-C2’/C6’), 125.6
(CF3), 111.7 (CH3=CH), 65.9 (CH-OH), 19.7 (CH3); ESI-MS m/z: 301.0 [M+H]+;
ESI-HRMS m/z: 301.068 [M+H]+, calcd for C14H12F3O4 301.0682.
5.1.1.5.
2-((4-Bromophenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-one
(1e)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-bromobenzaldehyde (439 mg, 2.40 mmol). The crude product
was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a white
solid 1e. (537 mg, 86.3%): mp 192.6-193.9 oC; 1H NMR (300 MHz, DMSO-d6) δ 2.18
(s, 3H, CH3), 5.98 (s, 1H, CH-OH), 6.20 (s, 1H, C=CH), 7.35 (d, J = 8.4 Hz, 2H,
Ph-H), 7.54 (d, J = 8.4 Hz, 2H, Ph-H); 13C NMR (75 MHz, DMSO-d6) δ 174.3 (C=O),
165.1 (CH3-C=CH), 150.4 (C-OH), 141.2 (Ar-C1’), 141.1 (HOCHC=COH), 131.6
(Ar-C3’/C5’), 128.6 (Ar-C2’/C6’), 120.9 (Ar-C4’), 111.6 (CH3=CH), 65.7 (CH-OH),
19.7 (CH3); ESI-MS m/z: 311.1 [M+H]+; ESI-HRMS m/z: 310.9914 [M+H]+, calcd
for C13H12BrO4 310.9913.

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5.1.1.6.
2-((3-Fluorophenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-one
(5a)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 3-fluorobenzaldehyde (298 mg, 2.40 mmol). The crude product
was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a white
solid 5a. (442 mg, 88.0%): mp 160.6-161.4 oC; 1H NMR (300 MHz, DMSO-d6)
(s, 3H, CH3), 6.02 (s, 1H, CH-OH), 6.20 (s, 1H, C=CH), 7.05-7.15 (m, 1H, Ph-H),
7.17-7.25 (m, 2H, Ph-H), 7.35-7.44 (m, 1H, Ph-H); 13C NMR (75 MHz, DMSO-d6)
δ 2.19
δ
174.4 (C=O), 165.1 (CH3-C=CH), 150.4 (C-OH), 144.9 (Ar-C1’), 144.8 (Ar-C3’)
141.3 (HOCHC=COH), 130.8 (Ar-C5’), 122.4 (Ar-C6’) , 114.7 (Ar-C4’), 113.2
(Ar-C2’), 111.6 (CH3=CH), 65.8 (CHArOH), 19.7 (CH3); ESI-MS m/z: 250.7 [M+H]+;
ESI-HRMS m/z: 251.0713 [M+H]+, calcd for C13H12FO4 251.0714.
5.1.1.7.
2-((3-Bromophenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-one
(5b)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 3-bromobenzaldehyde (440 mg, 2.40 mmol). The crude product
was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a white
o
1
solid 5b. (546 mg, 87.8%): mp 191.9-192.7 C; H NMR (300 MHz, DMSO-d6) δ
2.19 (s, 3H, CH3), 6.00 (s, 1H, CH-OH), 6.21 (s, 1H, C=CH), 7.29-7.39 (m, 2H,
Ph-H), 7.45-7.48 (m, 1H, Ph-H), 7.57-7.59 (m, 1H, Ph-H); 13C NMR (75 MHz,
DMSO-d6) δ 174.3 (C=O), 165.1 (CH3-C=CH), 150.3 (C-OH), 144.6 (Ar-C1’), 141.2
(HOCHC=COH), 131.0 (Ar-C2’), 130.7 (Ar-C4’), 129.0 (Ar-C5’), 125.4 (Ar-C6’),
122.1 (Ar-C3’), 111.7 (CH3=CH), 65.7 (CH-OH), 19.7 (CH3); ESI-MS m/z: 311.0
[M+H]+; ESI-HRMS m/z: 310.9914 [M+H]+, calcd for C13H12BrO4 310.9913.
5.1.1.8.
2-((2-fluorophenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-one
(5c)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 2-fluorobenzaldehyde (298 mg, 2.40 mmol). The crude product
was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a yellow
solid 5c. (457 mg, 82.1%): mp 165.5-166.7 oC; 1H NMR (400 MHz, DMSO-d6) δ 2.17
(s, 3H, CH3), 6.20 (s, 1H, CH-OH), 6.23 (s, 1H, C=CH), 7.13 (ddd, J = 10.6 Hz, J =
8.1 Hz, J = 1.2 Hz, 1H, Ph-H), 7.34 (tdd, J = 7.5 Hz, J = 5.3 Hz, J = 1.9 Hz, 1H,
Ph-H), 7.71 (td, J = 7.7 Hz, J = 1.9 Hz, 1H, Ph-H); 13C NMR (100 MHz, DMSO-d6) δ

ACCEPTED MANUSCRIPT
174.4 (C=O), 165.0 (CH3-C=CH), 160.6 (Ar-C2’) , 158.2 (Ar-C2’), 149.5 (C-OH),
141.2 (HOCHC=COH), 129.9 (Ar-C4’), 129.1 (Ar-C1’), 128.4 (Ar-C6’), 128.3
(Ar-C6’), 124.8 (Ar-C5’), 115.5 (Ar-C3’), 115.3 (Ar-C3’), 111.6 (CH3=CH), 60.8
(CH-OH), 19.7 (CH3); ESI-MS m/z: 250.6 [M+H]+; ESI-HRMS m/z: 251.0714
[M+H]+, calcd for C13H12FO4 251.0714.
5.1.1.9.
2-((2-Bromophenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-one
(5d)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 3-bromobenzaldehyde (440 mg, 2.40 mmol). The crude product
was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a yellow
o
solid 5d. (538 mg, 86.5%): mp 195.7-197.2 C; 1H NMR (400 MHz, DMSO-d6) δ
2.11 (s, 3H, CH3), 6.14 (s, 1H, CH-OH), 6.19 (s, 1H, C=CH), 7.25 (td, J = 7.6 Hz, J =
1.8 Hz, 1H, Ph-H), 7.46 (d, J = 7.6 Hz, J = 1.7 Hz, 1H, Ph-H), 7.56 (td, J = 8.0 Hz, J
= 1.2 Hz, 1H, Ph-H), 7.80 (d, J = 7.8 Hz, J = 1.2 Hz, 1H, Ph-H); 13C NMR (100 MHz,
DMSO-d6) δ 174.5 (C=O), 164.9 (CH3-C=CH), 148.9 (C-OH), 142.1 (Ar-C1’), 140.3
(HOCHC=COH), 132.7 (Ar-C3’), 129.9 (Ar-C4’), 129.8 (Ar-C6’), 128.0 (Ar-C5’),
121.7 (Ar-C2’), 111.6 (CH3=CH), 66.5 (CH-OH), 19.6 (CH3); ESI-MS m/z: 311.0
[M+H]+; ESI-HRMS m/z: 310.9916 [M+H]+, calcd for C13H12BrO4 310.9913.
5.1.1.10. 3-Hydroxy-2-(hydroxy(4-nitrophenyl)methyl)-6-methyl-4H-pyran-4-one (6a)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-nitrobenzaldehyde (363 mg, 2.40 mmol). The crude product
was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a yellow
solid 6a. (471 mg, 84.9%): mp 218.5-219.7 oC; 1H NMR (300 MHz, DMSO-d6)
δ 2.17
(s, 3H, CH3), 6.13 (s, 1H, CH-OH), 6.21 (s, 1H, C=CH), 6.49 (br.s, 1H, CH-OH), 7.67
(d, J = 8.6 Hz, 2H, Ph-H), 8.23 (d, J = 8.6 Hz, 2H, Ph-H), 9.33 (br.s, 1H, C=C-OH);
13C NMR (75 MHz, DMSO-d6)
δ 174.3 (C=O), 165.2 (CH3-C=CH), 149.7 (C-OH),
149.3 (Ar-C1’), 147.3 (Ar-C4’), 141.5 (HOCHC=COH), 127.6 (Ar-C2’/C6’), 124.0
(Ar-C3’/C5’), 111.7 (CH3=CH), 65.8 (CH-OH), 19.7 (CH3); ESI-MS m/z: 278.0
[M+H]+; ESI-HRMS m/z: 278.0657 [M+H]+, calcd for C13H12NO6 278.0659.
5.1.1.11.
3-Hydroxy-2-(hydroxy(4-(pentafluoro-λ6-sulfanyl)phenyl)methyl)-6-methyl-4H-pyran-
4-one (6b)

ACCEPTED MANUSCRIPT
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-(pentafluoro- λ6-sulfanyl)benzaldehyde (557 mg, 2.40 mmol).
The crude product was purified by column chromatography (MeOH:DCM=0.5:9.5) to
afford a light red solid 6b. (449 mg, 63.0%): mp 195.8-196.4 oC; 1H NMR (300 MHz,
DMSO-d6)
6.42 (d, J = 4.6 Hz, 1H, CH-OH), 7.62 (d, J = 8.6 Hz, 2H, Ph-H), 7.89 (d, J = 8.6 Hz,
2H, Ph-H), 9.29 (s, 1H, C=C-OH); 13C NMR (75 MHz, DMSO-d6)
174.3 (C=O),
δ 2.18 (s, 3H, CH3), 6.08 (d, J = 4.5 Hz, 1H, CH-OH), 6.21 (s, 1H, C=CH),
δ
165.2 (CH3-C=CH), 149.9 (C-OH), 146.4 (Ar-C1’), 141.4 (HOCHC=COH), 127.3
(Ar-C3’/C5’), 126.3 (Ar-C2’/C6’), 111.7 (CH3=CH), 65.7 (CH-OH), 19.7 (CH3);
ESI-MS m/z: 358.8 [M+H]+; ESI-HRMS m/z: 359.0370 [M+H]+, calcd for
C13H12F5O4S 359.0371.
5.1.1.12.
2-([1,1'-Biphenyl]-4-yl(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-one (6c)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and [1,1'-biphenyl]-4-carboxaldehyde (437 mg, 2.40 mmol). The
crude product was purified by column chromatography (MeOH:DCM=0.5:9.5) to
1
afford a yellow solid 6c. (501 mg, 81.3%): mp 187.4-188.2 oC; H NMR (300 MHz,
DMSO-d6)
δ 2.21 (s, 3H, CH3), 6.07 (s, 1H, CH-OH), 6.22 (s, 1H, C=CH), 7.32-7.38
(m, 1H, Ph-H), 7.45 (d, J = 7.7 Hz, 2H, Ph-H), 7.50 (d, J = 8.3 Hz, 2H, Ph-H), 7.50 (d,
13
J = 8.5 Hz, 4H, Ph-H), 9.20 (br.s, 1H, C=C-OH); C NMR (75 MHz, DMSO-d6)
δ
174.3 (C=O), 165.0 (CH3-C=CH), 150.9 (C-OH), 141.1 (HOCHC=COH), 140.3
(Ar-C1’/C4’’), 139.7 (Ar-C1’’), 129.4 (Ar-C1’/C5’/C2’’/C6’’), 127.9
(Ar-C2’/C6’/C3’’/C5’’), 127.1 (Ar-C4’), 111.6 (CH3=CH), 65.7 (CH-OH), 19.8 (CH3);
ESI-MS m/z: 309.1 [M+H]+; ESI-HRMS m/z: 309.1121 [M+H]+, calcd for C19H17O4
309.1121.
5.1.1.13.
3-Hydroxy-2-(hydroxy(4'-nitro-[1,1'-biphenyl]-4-yl)methyl)-6-methyl-4H-pyran-4-one
(6d)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4'-nitro-[1,1'-biphenyl]-4-carboxaldehyde (545 mg, 2.40 mmol).
The crude product was purified by column chromatography (MeOH:DCM=0.5:9.5) to
1
afford a yellow solid 6d. (511 mg, 72.2%): mp 184.6-185.7 oC; H NMR (300 MHz,
DMSO-d6) δ 2.20 (s, 3H, CH3), 6.08 (s, 1H, CH-OH), 6.21 (s, 1H, C=CH), 7.55 (d, J

ACCEPTED MANUSCRIPT
= 8.1 Hz, 2H, Ph-H), 7.79 (d, J = 8.1 Hz, 2H, Ph-H), 7.96 (d, J = 8.7 Hz, 2H, Ph-H),
7.96 (d, J = 8.7 Hz, 2H, Ph-H); 13C NMR (75 MHz, DMSO-d6) δ 174.3 (C=O), 165.1
(CH3-C=CH), 150.7 (C-OH), 147.1 (Ar-C4’), 146.8 (Ar-C1’), 142.8 (Ar-C4’’), 141.2
(HOCHC=COH), 137.3 (Ar-C1’’), 128.2 (Ar-C2’/C6’/C2’’/C6’’), 127.2
(Ar-C3’’/C5’’), 124.6 (Ar-C3’/C5’), 111.7 (CH3=CH), 65.0 (CH-OH), 19.7 (CH3);
ESI-MS m/z: 353.9 [M+H]+; ESI-HRMS m/z: 354.0971 [M+H]+, calcd for C19H16O6
354.0972.
5.1.1.14.
2-((4-(1H-imidazol-1-yl)phenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-on
e (6e)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-(1H-imidazol-1-yl)benzaldehyde (415 mg, 2.40 mmol). The
crude product was purified by column chromatography (MeOH:DCM=0.5:9.5) to
1
afford a yellow solid 6e. (422 mg, 70.4%): mp 183.8-184.3 oC; H NMR (300 MHz,
DMSO-d6) δ 2.20 (s, 3H, CH3), 6.06 (s, 1H, CH-OH), 6.21 (s, 1H, C=CH), 6.31 (br.s,
1H, CH-OH), 7.11 (s, 1H, Imidazole-H), 7.53 (d, J = 8.5 Hz, 2H, Ph-H), 7.64 (d, J =
8.5 Hz, 2H, Ph-H), 7.73 (s, 1H, Imidazole-H), 8.25 (s, 1H, Imidazole-H), 9.24 (br.s,
13
1H, C=C-OH); C NMR (75 MHz, DMSO-d6) δ 174.3 (C=O), 165.1 (CH3-C=CH),
150.7 (C-OH), 141.1 (HOCHC=COH), 140.5 (Ar-C1’’), 136.5 (Ar-C4’), 136.0
(Imidazole-C2’) , 130.2 (Imidazole-C4’), 127.8 (Ar-C2’’/C6’’), 120.8 (Ar-C3’’/C5’’),,
118.5 (Imidazole-C5’), 111.7 (CH3=CH), 65.7 (CH-OH), 19.7 (CH3); ESI-MS m/z:
299.0 [M+H]+; ESI-HRMS m/z: 299.1025 [M+H]+, calcd for C16H15N2O4 299.1026.
5.1.1.15. 2-((4-Benzylphenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-one
(6f)
The reaction was performed according to the general procedure A, using 4 (250 mg,
2.00 mmol) and 4-benzylbenzaldehyde (470 mg, 2.40 mmol). The crude product was
purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a yellow solid
1
6f. (458 mg, 71.0%): mp 169.7-170.6 oC; H NMR (300 MHz, DMSO-d6) δ 2.18 (s,
3H, CH3), 3.92 (s, 2H, Bn-H), 5.96 (s, 1H, CH-OH), 6.19 (s, 1H, C=CH), 7.10-7.35
13
(m, 9H, Ph-H); C NMR (75 MHz, DMSO-d6) δ 174.2 (C=O), 165.0 (CH3-C=CH),
151.0 (C-OH), 141.7 (Ar-C4’’/Bn-C1’), 140.9 (HOCHC=COH), 139.5 (Ar-C1’’),
129.1 (Ar-C3’’/C5’’), 129.0 (Bn-C3’’/C5’’), 128.9 (Bn-C2’’/C6’’), 126.6
(Ar-C2’’/C6’’), 126.4 (Bn-C4’), 111.6 (CH3=CH), 66.1 (CH-OH), 41.25 (Bn-CH2),

ACCEPTED MANUSCRIPT
19.7 (CH3); ESI-MS m/z: 323.1 [M+H]+; ESI-HRMS m/z: 323.1278 [M+H]+, calcd
for C20H19O4 323.1278.
5.1.1.16.
2-((4-(Benzyloxy)phenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-one (6g)
The reaction was performed according to the general procedure A, using 1 (1.00 g,
7.90 mmol) and 4-(benzyloxy)benzaldehyde (2.00 g, 9.48 mmol). The crude product
was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a white
solid 6g. (1.92 g, 63%): mp 180.2-180.7 oC; 1H NMR (300 MHz, DMSO-d6)
δ 2.20 (s,
3H, CH3), 5.08(s, 2H, OCH2-Ph), 6.06 (s, 2H, CH-OH), 6.19 (s, 1H, C=CH), 6.99 (d,
13
J = 8.4 Hz, 2H, Ph-H), 7.31-7.45 (m, 7H, Ph-H); C NMR (75 MHz, DMSO-d6)
δ
174.3 (C=O), 164.9 (CH3-C=CH), 158.1 (Ar-C4’’), 151.2 (C-OH), 140.8
(HOCHC=COH), 137.5 (Ph-C1’), 134.1 (Ar-C1’’), 128.9 (Ph-C3’/C5’), 128.1
(Ar-C2’’/C6’’), 127.7 (Ph-C2’/C6’), 115.0 (Ar-C3’’/C5’’), 111.5 (CH3=CH), 69.6
(Bn-CH2), 66.0 (CH-OH), 19.7 (CH3); ESI-MS m/z: 339.1 [M+H]+；ESI-HRMS m/z:
339.1226 [M+H]+, calcd for C20H19O5 339.1227.
5.1.1.17.
3-Hydroxy-2-(hydroxy(4-((4-nitrobenzyl)oxy)phenyl)methyl)-6-methyl-4H-pyran-4-on
e (6h)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-((4-nitrobenzyl)oxy)benzaldehyde [26] (620 mg, 2.40 mmol).
The crude product was purified by column chromatography (MeOH:DCM=0.5:9.5) to
1
afford a yellow solid 6h. (454 mg, 58.7%): mp 178.8-188.7 oC; H NMR (300 MHz,
DMSO-d6) δ 2.19 (s, 3H, CH3), 5.27 (s, 2H, CH2-Ph)，5.94 (d，J = 4.8 Hz, 1H,
CH-OH), 5.08 (d, J = 4.8 Hz, 1H, CH-OH), 6.19 (s, 1H, C=CH), 7.01 (d, J = 8.7 Hz,
2H, Ph-H), 7.34 (d, J = 8.6 Hz, 2H, Ph-H), 7.70 (d, J = 8.6 Hz, 2H, Ph-H), 8.25 (d, J =
8.7 Hz, 2H, Ph-H), 9.07 (br.s, 1H, C=C-OH); 13C NMR (75 MHz, DMSO-d6) δ 174.2
(C=O), 165.9 (CH3-C=CH), 157.7 (Ar-C4’’), 151.1 (C-OH), 147.4 (Ar-C4’), 145.5
(Ar-C1’), 140.8 (HOCHC=COH), 134.4 (Ar-C1’’), 128.6 (Ar-C2’/C6’), 127.8
(Ar-C2’’/C6’’), 124.1 (Ar-C3’/C5’), 115.0 (Ar-C3’’/C5’’), 111.6 (CH3=CH), 68.4
(Ar-CH2), 65.9 (CH-OH), 19.7 (CH3); ESI-MS m/z: 384.0 [M+H]+；ESI-HRMS m/z:
384.1076 [M+H]+, calcd for C20H18NO7 384.1078.
5.1.1.18. 3-Hydroxy-2-(hydroxy(4-phenethylphenyl)methyl)-6-methyl-4H-pyran-4-one
(6i)

ACCEPTED MANUSCRIPT
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-phenethylbenzaldehyde [27] (420 mg, 2.40 mmol). The crude
product was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a
o
1
light red solid 6i. (486mg, 72.3%): mp 180.6-181.2 C; H NMR (300 MHz,
DMSO-d6) 2.19 (s, 3H, CH3), 2.86 (s, 4H, Ph-(CH2)2-Ph)，5.97 (d，J = 4.9 Hz, 1H,
δ
CH-OH), 5.09 (d, J = 4.9 Hz, 1H, CH-OH), 6.19 (s, 1H, C=CH), 7.15-7.35 (m, 9H,
13
Ph-H), 9.08 (s, 1H, C=C-OH); C NMR (75 MHz, DMSO-d6)
δ
174.3 (C=O), 164.9
(CH3-C=CH), 151.10 (C-OH), 142.0 (Ar-C4’’/Ph-C1’), 140.9 (HOCHC=COH), 139.3
(Ar-C1’’), 128.7 (Ar-C3’’/C5’’, Ph-C3’/C5’), 126.4 (Ar-C2’’/C6’’, Ph-C2’/C6’), 111.6
(CH3=CH), 66.2 (CH-OH), 37.5 (Ar-CH2), 37.2 (Ph-CH2), 19.7 (CH3); ESI-MS m/z:
337.1 [M+H]+；ESI-HRMS m/z: 337.1430 [M+H]+, calcd for C21H21O4 337.1434.
5.1.1.19. (E)-3-Hydroxy-2-(hydroxy(4-styrylphenyl)methyl)-6-methyl-4H-pyran-4-one
(6j)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and (E)-4-styrylbenzaldehyde (501 mg, 2.40 mmol). The crude
product was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a
o
1
light red solid 6j. (466mg, 67.6%): mp 180.1-180.9 C; H NMR (300 MHz,
DMSO-d6) δ 2.20 (s, 3H, CH3), 6.02 (d，J = 4.4 Hz, 1H, CH-OH), 6.18 (d, J = 4.4 Hz,
1H, CH-OH), 6.21 (s, 1H, C=CH), 7.24 (s, 2H, CH=CH), 7.28 (d，J = 7.2 Hz, 1H,
Ph-H), 7.36 (d, J = 7.5 Hz, 2H, Ph-H), 7.42 (d, J = 8.5 Hz, 2H, Ph-H), 7.60 (d, J = 9.0
Hz, 4H, Ph-H), 9.13 (s, 1H, C=C-OH); 13C NMR (75 MHz, DMSO-d6) δ 174.3 (C=O),
165.0 (CH3-C=CH), 150.9 (C-OH), 141.2 (Ar-C1’’), 141.0 (HOCHC=COH), 137.5
(Ar-C4’’) , 136.7 (Ph-C1’), 129.2 (Ar-C3’’/C5’’), 128.8 (Ph-C2’/C3’/C5’/C6’), 128.5
(Ph-C4’), 128.1 (Ar-C2’’/C6’’), 126.9 (CH=CH), 126.8 (Ar-C2’’/C6’’), 111.6
(CH3=CH), 66.2 (CH-OH), 19.7 (CH3); ESI-MS m/z: 335.1 [M+H]+；ESI-HRMS m/z:
335.1272 [M+H]+, calcd for C21H19O4 335.1278.
5.1.1.20.
3-Hydroxy-2-(hydroxy(4-(phenylethynyl)phenyl)methyl)-6-methyl-4H-pyran-4-one
(6k)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-(phenylethynyl)benzaldehyde (495 mg, 2.40 mmol). The crude
product was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a
o
1
light red solid 6k. (492mg, 70.0%): mp 188.8-189.9 C; H NMR (300 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6) δ 2.19 (s, 3H, CH3), 6.04 (s, 1H, CH-OH), 6.21 (s, 1H, C=CH), 6.28 (bs.s,
1H, CH-OH), 7.24 (s, 2H, CH=CH), 7.40-7.46 (m, 5H, Ph-H), 7.54-7.57 (m, 4H,
Ph-H), 9.21 (s, 1H, C=C-OH); 13C NMR (75 MHz, DMSO-d6) δ 174.3 (C=O), 165.1
(CH3-C=CH), 150.5 (C-OH), 142.5 (Ar-C1’’), 141.2 (HOCHC=COH), 131.8
(Ar-C3’’/C5’’, Ph-C2’/C6’), 129.2 (Ph-C), 126.8 (Ar-C2’’/C6’’), 122.7 (Ar-C), 121.7
(Ar-C4’’), 111.7 (CH3=CH), 89.7 (Alkyne-C), 66.0 (CH-OH), 19.70 (CH3); ESI-MS
m/z: 333.0 [M+H]+；ESI-HRMS m/z: 333.1120 [M+H]+, calcd for C21H17O4 333.1121.
5.1.1.21.
2-((4-((4-Fluorophenyl)ethynyl)phenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyra
n-4-one (6l)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-((4-fluorophenyl)ethynyl)benzaldehyde (539 mg, 2.40 mmol).
The crude product was purified by column chromatography (MeOH:DCM=0.5:9.5) to
afford a light red solid 6l. (504 mg, 72.0%): mp 205.9-206.6 oC; 1H NMR (300 MHz,
DMSO-d6) δ 2.19 (s, 3H, CH3), 6.04 (d，J = 4.9 Hz, 1H, CH-OH), 6.21 (s, 1H,
C=CH), 6.27 (d, J = 4.9 Hz, 1H, CH-OH), 7.24 (s, 2H, CH=CH), 7.28 (d，J = 7.2 Hz,
1H, Ph-H), 7.36 (d, J = 7.5 Hz, 2H, Ph-H), 7.27 (t, J = 8.8 Hz, 2H, Ph-H), 7.46 (d, J =
8.2 Hz, 2H, Ph-H), 7.55 (d, J = 8.2 Hz, 2H, Ph-H), 7.62 (dd, J = 8.6 Hz, J = 5.5 Hz,
13
2H, Ph-H), 9.20 (s, 1H, C=C-OH); C NMR (75 MHz, DMSO-d6) δ 174.3 (C=O),
165.1 (CH3-C=CH), 160.8 (Ar-C4’), 150.5 (C-OH), 142.5 (Ar-C1’’), 141.2
(HOCHC=COH), 131.8 (Ar-C3’’/C5’’, Ph-C2’/C6’), 126.8 (Ar-C2’’/C6’’), 121.6
(Ar-C4’’), 119.16 (Ar-C1’), 116.3 (Ar-C3’/C5’), 111.6 (CH3=CH), 88.7 (Alkyne-C),
66.1 (CH-OH), 19.7 (CH3); ESI-MS m/z: 351.0 [M+H]+；ESI-HRMS m/z: 351.1025
[M+H]+, calcd for C21H16FO4 351.1027.
5.1.1.22.
2-((4-((3-Fluorophenyl)ethynyl)phenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyra
n-4-one (6m)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-((3-fluorophenyl)ethynyl)benzaldehyde (539 mg, 2.40 mmol).
The crude product was purified by column chromatography (MeOH:DCM=0.5:9.5) to
afford a light red solid 6m. (512 mg, 73.0%): mp 189.6-190.2 oC; 1H NMR (300 MHz,
DMSO-d6)
δ 2.19 (s, 3H, CH3), 6.04 (d, J =4.8Hz, 1H, CH-OH), 6.21 (s, 1H, C=CH),
6.28 (d, J =4.9Hz, 1H, CH-OH), 7.24-7.32 (m, 1H, Ph-H), 7.38-7.50 (m, 5H, Ph-H),

ACCEPTED MANUSCRIPT
7.57 (d, J =8.1Hz, 2H, Ph-H), 9.21 (s, 1H, C=C-OH); 13C NMR (75 MHz, DMSO-d6)
δ
174.3 (C=O), 165.1 (CH3-C=CH), 160.7 (Ar-C4’), 150.5 (C-OH), 142.9 (Ar-C1’’),
141.2(HOCHC=COH), 131.9 (Ar-C3’’/C5’’), 128.2 (Ar-C5’), 126.8 (Ar-C2’’/C6’’),
124.8 (Ar-C1’), 121.2 (Ar-C4’’), 118.2 (Ar-C2’), 116.6 (Ar-C4’), 111.6 (CH3=CH),
90.6 (Alkyne-C), 88.4 (Alkyne-C), 66.0 (CH-OH), 19.7 (CH3); ESI-MS m/z: 350.9
[M+H]+；ESI-HRMS m/z: 351.1028 [M+H]+, calcd for C21H16FO4 351.1027.
5.1.1.23.
3-Hydroxy-2-(hydroxy(4-((4-(trifluoromethyl)phenyl)ethynyl)phenyl)methyl)-6-methyl
-4H-pyran-4-one (6n)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-((4-(trifluoromethyl)phenyl)ethynyl)benzaldehyde (559 mg,
2.40 mmol). The crude product was purified by column chromatography
(MeOH:DCM=0.5:9.5) to afford a white solid 6n. (502 mg, 62.8%): mp 210.1-211.7
oC; 1H NMR (300 MHz, DMSO-d6) δ 2.19 (s, 3H, CH3), 6.05 (s, 1H, CH-OH), 6.21 (s,
1H, C=CH), 7.48 (d, J = 7.9 Hz, 2H, Ph-H), 7.60 (d, J = 8.0 Hz, 2H, Ph-H), 7.78 (s,
4H, Ph-H); 13C NMR (75 MHz, DMSO-d6) δ 174.3 (C=O), 165.1 (CH3-C=CH), 150.4
(C-OH), 143.1 (Ar-C1’’), 141.3 (HOCHC=COH), 132.6 (Ar-C2’/C6’), 132.1
(Ar-C3’’/C5’’), 126.0 (Ar-C2’’/C6’’, Ar-C1’), 122.6 (CF3), 121.0 (Ar-C4’’), 111.6
(CH3=CH), 92.2 (Alkyne-C), 88.3 (Alkyne-C), 66.1 (CH-OH), 19.7 (CH3); ESI-MS
m/z: 401.1 [M+H]+; ESI-HRMS m/z: 401.0996 [M+H]+, calcd for C22H16F3O4
401.0995.
5.1.1.24.
3-Hydroxy-2-(hydroxy(4-((4-methoxyphenyl)ethynyl)phenyl)methyl)-6-methyl-4H-pyr
an-4-one(6o)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and4-((4-methoxyphenyl)ethynyl)benzaldehyde (568 mg, 2.40 mmol).
The crude product was purified by column chromatography (MeOH:DCM=0.5:9.5) to
o
1
afford a white solid 6o. (494 mg, 68.2%): mp 192.2-193.2 C; H NMR (300 MHz,
DMSO-d6) δ 2.19 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 6.03 (d, J = 4.8 Hz, 1H, CH-OH),
6.21 (s, 1H, C=CH), 6.25 (d, J = 4.9 Hz, 1H, CH-OH), 6.98 (d, J = 8.7 Hz, 2H, Ph-H),
7.43 (d, J = 8.2 Hz, 2H, Ph-H), 7.78 (t, J = 8.0 Hz, 4H, Ph-H), 9.18 (s, 1H, C=C-OH);
13C NMR (75 MHz, DMSO-d6) δ 174.3 (C=O), 165.1 (CH3-C=CH), 160.0 (Ar-C4’),
150.5 (C-OH), 142.1 (Ar-C1’’), 141.2 (HOCHC=COH), 133.4 (Ar-C3’’/C5’’), 131.6

ACCEPTED MANUSCRIPT
(Ar-C2’/C6’), 126.7 (Ar-C2’’/C6’’), 122.1 (Ar-C4’’), 114.9 (Ar-C3’/C5’), 111.6
(CH3=CH), 89.1 (Alkyne-C), 88.3 (Alkyne-C), 66.1 (CH-OH), 55.7 (OCH3), 19.7
(CH3); ESI-MS m/z: 363.1 [M+H]+; ESI-HRMS m/z: 363.1228 [M+H]+, calcd for
C22H19O5 363.1227.
5.1.1.25.
2-((4-(Cyclohexylethynyl)phenyl)(hydroxy)methyl)-3-hydroxy-6-methyl-4H-pyran-4-o
ne (6p)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-(cyclohexylethynyl)benzaldehyde (511 mg, 2.40 mmol). The
crude product was purified by column chromatography (MeOH:DCM=0.5:9.5) to
1
afford a white solid 6p. (484 mg, 71.5%): mp 199.4-200.1 oC; H NMR (300 MHz,
DMSO-d6) δ 1.30-1.53 (m, 6H, Cyclohexane-H), 1.60-1.72 (m, 2H, Cyclohexane-H ),
1.78-1.84 (m, 2H, Cyclohexane-H ), 2.19 (s, 3H, CH3), 2.58-2.66 (m, 1H,
Cyclohexane-H ), 6.00 (d, J = 3.9 Hz, 1H, CH-OH), 6.21 (s, 2H, C=CH, CH-OH),
13
6.98 (d, J = 8.7Hz, 2H, Ph-H), 7.36 (s, 4H, Ph-H), 9.13 (s, 1H, C=C-OH); C NMR
(75 MHz, DMSO-d6)
δ 174.3 (C=O), 165.0 (CH3-C=CH), 150.6 (C-OH), 141.5
(Ar-C1’’), 141.1 (HOCHC=COH), 131.6 (Ar-C3’’/C5’’), 126.5 (Ar-C2’’/C6’’), 122.7
(Ar-C4), 111.6 (CH3=CH), 94.8 (Alkyne-C), 80.8 (Alkyne-C), 66.0 (CH-OH), 32.7
(Cyclohexane-C2’/C6’), 29.2 (Cyclohexane-C1’), 25.8 (Cyclohexane-C4’’), 24.7
(Cyclohexane-C3’/C5’), 19.7 (CH3); ESI-MS m/z: 339.1 [M+H]+; ESI-HRMS m/z:
339.1590 [M+H]+, calcd for C21H23O4 339.1591.
5.1.1.26.
3-Hydroxy-2-(hydroxy(4-(pyridin-2-ylethynyl)phenyl)methyl)-6-methyl-4H-pyran-4-on
e (6q)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 4-(pyridin-2-ylethynyl)benzaldehyde (497 mg, 2.40 mmol). The
crude product was purified by column chromatography (MeOH:DCM=0.5:9.5) to
1
afford a white solid 6q. (467 mg, 70.5%): mp 169.3-170.1oC; H NMR (300 MHz,
DMSO-d6)
δ 2.19 (s, 3H, CH3), 6.05 (s, 1H, CH-OH), 6.21 (s, 1H, C=CH), 7.43-7.50
(m, 3H, Ph-H, Pyridine-H), 7.57-7.60 (m, 2H, Ph-H), 7.95-7.99 (m, 1H, Pyridine-H),
13
8.57-8.60 (m, 1H, Pyridine-H), 8.75-8.76 (m, 1H, Pyridine-H); C NMR (75 MHz,
DMSO-d6)
δ 174.4 (C=O), 165.0 (CH3-C=CH), 152.0, 150.5 (C-OH), 149.4
(Pyridine-C6’), 143.1 (Pyridine-C2’), 141.4 (HOCHC=COH), 139.0 (Pyridine-C4’),
131.9 (Ar-C3’’/C5’’), 126.8 (Ar-C2’’/C6’’), 124.1 (Pyridine-C5’), 121.1 (Ar-C4’’),

ACCEPTED MANUSCRIPT
119.9, 111.6 (CH3=CH), 92.7 (Alkyne-C), 86.6 (Alkyne-C), 66.1 (CH-OH), 19.7
(CH3); ESI-MS m/z: 333.9 [M+H]+; ESI-HRMS m/z: 334.1074 [M+H]+, calcd for
C20H26O4 334.1074.
5.1.1.27. 3-Hydroxy-2-(1-hydroxybutyl)-6-methyl-4H-pyran-4-one (7a)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and butyraldehyde (174 mg, 2.40 mmol). The crude product was
purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a light red solid
7a. (304 mg, 76.5%): mp 125.3-126.0 oC; 1H NMR (300 MHz, DMSO-d6) δ 0.87 (t, J
= 7.3 Hz, 3H, -CH2CH3), 1.15-1.40 (m, 2H, -CH2CH3), 1.51-1.79 (m, 2H,
-CHOHCH2), 2.26 (s, 3H, CH3), 4.80 (t, J = 7.3 Hz, 1H, -CHOHCH2), 5.34 (s, 1H,
-CHOH), 6.20 (s, 1H, C=CH), 8.77 (s, 1H, C=C-OH); 13C NMR (75 MHz, DMSO-d6)
δ 174.2 (C=O), 164.9 (CH3-C=CH), 151.7 (C-OH), 140.9 (HOCHC=COH), 111.4
(CH3=CH), 64.3 (CH-OH), 36.5 (CHOH-CH2), 19.7 (CH=C-CH3), 18.8 (CH2-CH3),
14.2 (CH2-CH3); ESI-MS m/z: 199.0 [M+H]+; ESI-HRMS m/z: 199.0965 [M+H]+,
calcd for C10H15O4 199.0965.
5.1.1.28. 3-Hydroxy-2-(1-hydroxypentyl)-6-methyl-4H-pyran-4-one (7b)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and pentanal (207 mg, 2.40 mmol). The crude product was purified
by column chromatography (MeOH:DCM=0.5:9.5) to afford a light red solid 7b. (323
mg, 76.1%): mp 123.8-124.8 oC; 1H NMR (300 MHz, DMSO-d6)
δ 0.85 (t, J = 7.1 Hz,
3H, -CH2CH3), 1.10-1.42 (m, 4H, -(CH2)2CH3), 1.52-1.80 (m, 2H, -CHOHCH2), 2.26
(s, 3H, CH3), 4.78 (td, J = 7.3 Hz,J = 5.0 Hz, 1H, -CHOHCH2), 5.33 (d, J = 5.0 Hz,
1H, -CHOH), 6.20(s, 1H, C=CH), 8.74 (s, 1H, C=C-OH); 13C NMR (75 MHz,
DMSO-d6)
δ
174.2 (C=O), 164.9 (CH3-C=CH), 151.7 (C-OH), 141.0
(HOCHC=COH), 111.4 (CH3=CH), 64.6 (CH-OH), 34.1 (CHOH-CH2), 27.7 (CH2),
22.35 (CH2-CH3), 19.7 (CH=C-CH3), 14.4 (CH2-CH3); ESI-MS m/z: 213.2 [M+H]+;
ESI-HRMS m/z: 213.1124 [M+H]+, calcd for C11H17O4 213.1121.
5.1.1.29. 3-Hydroxy-2-(1-hydroxyhexyl)-6-methyl-4H-pyran-4-one (7c)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and hexanal (241 mg, 2.40 mmol). The crude product was purified by
column chromatography (MeOH:DCM=0.5:9.5) to afford a light red solid 7c. (316
mg, 70.1%): mp 122.5-123.4 oC; 1H NMR (300 MHz, DMSO-d6) δ 0.84 (t, J = 6.5 Hz,
3H, -CH2CH3), 1.15-1.37 (m, 6H, -(CH2)3CH3), 1.54-1.78 (m, 2H, -CHOHCH2), 2.26

ACCEPTED MANUSCRIPT
(s, 3H, CH3), 4.78 (t, J = 7.1 Hz, 1H, -CHOHCH2), 5.33 (s, 1H, -CHOH), 6.20 (s, 1H,
C=CH), 8.74 (s, 1H, C=C-OH); 13C NMR (75 MHz, DMSO-d6) δ 174.2 (C=O), 164.8
(CH3-C=CH), 151.7 (C-OH), 141.0 (HOCHC=COH), 111.4 (CH3=CH), 64.6
(CH-OH), 34.4 (CHOH-CH2), 31.4 (CH2), 25.1 (CH2), 22.5 (CH2-CH3), 19.7
(CH=C-CH3), 14.3 (CH2-CH3); ESI-MS m/z: 227.1 [M+H]+; ESI-HRMS m/z:
227.1282 [M+H]+, calcd for C12H19O4 227.1278.
5.1.1.30. 3-Hydroxy-2-(1-hydroxyheptyl)-6-methyl-4H-pyran-4-one (7d)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and heptanal (274 mg, 2.40 mmol). The crude product was purified
by column chromatography (MeOH:DCM=0.5:9.5) to afford a light red solid 7d. (345
mg, 71.8%): mp 120.6-121.4 oC; 1H NMR (300 MHz, DMSO-d6) δ 0.82 (t, J = 6.8 Hz,
3H, -CH2CH3), 1.15-1.37 (m, 8H, -(CH2)4CH3), 1.54-1.78 (m, 2H, -CHOHCH2), 2.26
(s, 3H, CH3), 4.78 (t, J = 5.7 Hz, 1H, -CHOHCH2), 5.33 (s, 1H, -CHOH), 6.20 (s, 1H,
C=CH), 8.73 (s, 1H, C=C-OH); 13C NMR (75 MHz, DMSO-d6) δ 174.2 (C=O), 164.8
(CH3-C=CH), 151.7 (C-OH), 141.0 (HOCHC=COH), 111.4 (CH3=CH), 64.6
(CH-OH), 34.4 (CHOH-CH2), 31.6 (CH2), 28.9 (CH2), 25.4 (CH2), 22.5 (CH2-CH3),
19.7 (CH=C-CH3), 14.4 (CH2-CH3); ESI-MS m/z:241.1 [M+H]+; ESI-HRMS m/z:
241.1434 [M+H]+, calcd for C13H21O4 241.1434.
5.1.1.31. 3-Hydroxy-2-(1-hydroxyoctyl)-6-methyl-4H-pyran-4-one (7e)
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and octanal (308 mg, 2.40 mmol). The crude product was purified by
column chromatography (MeOH:DCM=0.5:9.5) to afford a light red solid 7e. (305
mg, 60.0%): mp 120.1-120.9 oC; 1H NMR (300 MHz, DMSO-d6)
δ 0.85 (t, J = 6.8 Hz,
3H, -CH2CH3), 1.15-1.37 (m, 10H, -(CH2)5CH3), 1.54-1.79 (m, 2H, -CHOHCH2),
2.26 (s, 3H, CH3), 4.78 (td, J = 6.8 Hz, J = 5.1 Hz, 1H, -CHOHCH2), 5.33 (s, J = 5.1
13
Hz, 1H, -CHOH), 6.20 (s, 1H, C=CH), 8.74 (s, 1H, C=C-OH); C NMR (75 MHz,
DMSO-d6)
δ
174.2 (C=O), 164.8 (CH3-C=CH), 151.7 (C-OH), 141.0
(HOCHC=COH), 111.4 (CH3=CH), 64.6 (CH-OH), 34.4 (CHOH-CH2), 31.7 (CH2),
29.2 (CH2), 29.1 (CH2), 25.5 (CH2), 22.5 (CH2-CH3), 19.7 (CH=C-CH3), 14.4
(CH2-CH3); ESI-MS m/z:255.2 [M+H]+; ESI-HRMS m/z: 255.1591 [M+H]+, calcd
for C13H23O4 255.1591.
5.1.1.32. 3-Hydroxy-2-(1-hydroxy-3-(methylthio)propyl)-6-methyl-4H-pyran-4-one
(7f)

ACCEPTED MANUSCRIPT
The reaction was performed according to the general procedure A, using 4 (250
mg, 2.00 mmol) and 3-(methylthio)propanal (250 mg, 2.40 mmol). The crude product
was purified by column chromatography (MeOH:DCM=0.5:9.5) to afford a light red
solid 7f. (325 mg, 70.6%): mp 124.1-125.2 oC; 1H NMR (300 MHz, DMSO-d6) δ 0.84
(t, J = 6.5 Hz, 3H, -CH2CH3), 1.52-2.0 (m, 2H, -CH2-S), 2.04 (s, 3H, CH3-S), 2.26 (s,
3H, CH3), 2.46-2.58 (m, 2H, CH2-CH2S), 4.85-4.95 (m, 1H, CH-OH), 5.50 (d, J = 8.0
13
Hz, 1H, -CHOH), 6.21 (s, 1H, C=CH), 8.85 (s, 1H, C=C-OH); C NMR (75 MHz,
DMSO-d6)
δ
174.2 (C=O), 165.0 (CH3-C=CH), 151.2 (C-OH), 141.0
(HOCHC=COH), 111.5 (CH3=CH), 63.5 (CH-OH), 33.9 (CHOH-CH2), 29.8 (CH2),
19.8 (CH=C-CH3), 15.1 (S-CH3)); ESI-MS m/z:231.1 [M+H]+; ESI-HRMS m/z:
231.0686 [M+H]+, calcd for C10H15O4S 231.0683.
5.1.2. General procedure B for the reaction of 8a-8d
After dissolving 1.0 equiv of compounds 1a-1d in dry DCM, 5.0 equiv of
triethylsilane was added at 0 oC. The homogeneous mixture was treated with 5.0 equiv
o
of trifluoroacetic acid, and stirred at 0 C for 45 min then at room temperature
overnight. The reaction mixture were extracted with DCM, and dried over anhydrous
MgSO4, provided the crude product which was purified by chromatography [28].
5.1.2.1. 2-Benzyl-3-hydroxy-6-methyl-4H-pyran-4-one (8a)
According to procedure B, 1a (1.16 g, 5.00 mmol) was treated with triethylsilane
(2.91 g, 25.00 mmol). The crude product was purified by chromatography to provide
1.00 g (92.2%) of 8a as white solid: mp 141.6-142.7 oC; 1H NMR (300 MHz, CDCl3)
δ 2.26 (s, 3H, CH3), 4.03 (s, 2H, CH2-Ph), 6.22 (s, 1H, C=CH), 7.26-7.34 (m, 5H,
Ph-H); 13C NMR (75 MHz, CDCl3) δ 174.5 (C=O), 165.4 (CH3-C=CH), 149.4
(C-OH), 141.9 (HOCHC=COH), 136.5 (Ph-C1’), 128.8 (Ph-C2’/C6’), 128.7
(Ph-C3’/C5’), 126.9 (Ph-C4’), 110.79 (CH3=CH), 34.4 (Ar-CH2), 20.1 (CH=C-CH3);
ESI-MS m/z: 217.0 [M+H]+; ESI-HRMS m/z: 217.0857 [M+H]+, calcd for C13H13O3
217.0859.
5.1.2.2. 2-(4-Fluorobenzyl)-3-hydroxy-6-methyl-4H-pyran-4-one (8b)
According to procedure B, 1b (1.25 g, 5.00 mmol) was treated with triethylsilane
(2.91 g, 25.00 mmol). The crude product was purified by chromatography to provide
1.10 g (94.5%) of 8b as white solid: mp 158.7-159.6 oC; 1H NMR (300 MHz, CDCl3)
δ 2.26 (s, 3H, CH3), 4.00 (s, 2H, CH2-Ph), 6.22 (s, 1H, C=CH), 7.00 (t, J = 8.7 Hz, 2H,
13
Ph-H), 7.27 (dd, J = 8.7 Hz, J = 6.4 Hz, 2H, Ph-H); C NMR (75 MHz, CDCl3) δ

ACCEPTED MANUSCRIPT
174.2 (C=O), 165.4 (CH3-C=CH), 160.3 (Ar-C4’), 149.2 (C-OH), 141.4
(HOCHC=COH), 132.2 (Ar-C1’), 130.3 (Ar-C2’/C6’), 115.6 (Ar-C3’/C5’), 110.8
(CH3=CH), 33.6 (Ar-CH2), 20.0 (CH=C-CH3); ESI-MS m/z: 235.0 [M+H]+;
ESI-HRMS m/z: 235.0757 [M+H]+, calcd for C13H12FO3 235.0765.
5.1.2.3. 2-(4-Chlorobenzyl)-3-hydroxy-6-methyl-4H-pyran-4-one (8c)
According to procedure B, 1c (1.33 g, 5.00 mmol) was treated with triethylsilane
(2.91 g, 25.00 mmol). The crude product was purified by chromatography to provide
1.24 g (92.8%) of 8c as white solid: mp 170.1-171.0 oC; 1H NMR (300 MHz, CDCl3)
δ 2.25 (s, 3H, CH3), 3.99 (s, 2H, CH2-Ph), 6.22 (s, 1H, C=CH), 7.23-7.30 (m, 4H,
Ph-H); 13C NMR (75 MHz, CDCl3) δ 174.2 (C=O), 165.4 (CH3-C=CH), 149.1
(C-OH), 141.6 (HOCHC=COH), 135.0 (Ar-C1’), 132.8 (Ar-C4’), 130.20
(Ar-C2’/C6’), 128.8 (Ar-C3’/C5’), 110.9 (CH3=CH), 33.7 (Ar-CH2), 20.0
(CH=C-CH3); ESI-MS m/z: 251.0 [M+H]+; ESI-HRMS m/z: 251.0460 [M+H]+, calcd
for C13H12ClO3 251.0469.
5.1.2.4. 3-Hydroxy-6-methyl-2-(4-(trifluoromethyl)benzyl)-4H-pyran-4-one (8d)
According to procedure B, 1d (1.50 g, 5.00 mmol) was treated with triethylsilane
(2.91 g, 25.00 mmol). The crude product was purified by chromatography to provide
1.35 g (90.4%) of 8d as white solid: mp 159.8-161.0 oC; 1H NMR (300 MHz, CDCl3)
δ 2.27 (s, 3H, CH3), 4.08 (s, 2H, CH2-Ph), 6.24 (s, 1H, C=CH), 7.43 (d, J = 8.1 Hz,
2H, Ph-H), 7.57 (d, J = 8.1 Hz, 2H, Ph-H); 13C NMR (75 MHz, CDCl3) δ 174.1
(C=O), 165.5 (CH3-C=CH), 148.5 (C-OH), 141.7 (HOCHC=COH), 140.6 (Ar-C1’),
129.2 (Ar-C2’/C6’), 125.6 (Ar-C3’/C5’), 122.3 (Ar-C4’), 111.0 (CF3), 34.18 (Ar-CH2),
20.02 (CH=C-CH3); ESI-MS m/z: 285.0 [M+H]+; ESI-HRMS m/z: 285.0731 [M+H]+,
calcd for C14H12F3O3 285.0733.
5.2. Biological Assays
5.2.1 MIC determination and growth curve assay
A broth-microdilution method from standards published by the Clinical and
Laboratory Standards Institute [29] was employed to determine the minimum
inhibitory concentrations (MIC) of compounds. Briefly, two-fold serial dilutions of
compounds were prepared in LB medium, at a volume of 100 µL per well in 96-well
U-bottomed polystyrene microtiter plates (Corning/Costar, NY, USA). Each well was
inoculated with 100 µL of the standardized P. aeruginosa PAO1 inoculum, yielding a
final bacterial concentration of approximately 1 × 107 CFU/mL. The final tested

ACCEPTED MANUSCRIPT
concentrations of antimicrobial agents ranged from 0-521 µM for compounds. To
construct growth curves, overnight cultures of P. aeruginosa PAO1 inoculum were
diluted in 100 µL of fresh LB broth to achieve a cell suspension optical density at 600
nm (OD600) of 0.05. The growth curve was recorded every 10 min with Tecan Infinate
200 Pro plate reader (Tecan Group Ltd., Männedorf, Switzerland) for 15 h.
5.2.2. Biofilm assessment
Involvement of compounds in biofilm formation was determined by a Crystal
o
Violet assay [30]. P. aeruginosa PAO1 was grown overnight in LB medium at 37 C
for 24 h. The evaluation of biofilm formation used sterile 96-well flat-bottomed
polystyrene microtiter plates (Corning/Costar, NY, USA) and a culture of 100 µl of
o
bacterial (1 × 107 CFU/mL) in ABTGC medium at 37 C for 24 h. Subsequently,
supernatant cells were removed and the biofilms were washed three times with sterile
PBS, and subsequently with MeOH as a fixative. After 30 min, the MeOH was
removed, and the microtiter plates were dried at room temperature. Crystal Violet (0.1%
in water) was then added to dye the biofilms and the mixture was incubated for 30
min. The Crystal Violet was removed, the microtiter plates were rinsed three times
with sterile PBS and 150 µl of AcOH (33%) was added to dissolve the biofilm. The
absorbance values were measured at 570 nm by a microplate reader. All compounds
were dissolved in DMSO to make 100 mM stock solutions before being transferred to
96-well plates for the experiment.
5.2.3. Elastase Assay.
An overnight culture of P. aeruginosa PAO1 in 5 mL of LB medium was diluted
to a final optical density at 600 nm (OD600) of 0.01. The culture was incubated with
different concentrations of compounds (40, 20, 10, 5, 2.5 and 0 µM) for 24 h at 37 oC，
with shaking at 200 rpm. The supernatant was collected after centrifugation at 10000
rpm for 7 min. Elastase activity was measured with an EnzChekElastase assay kit
(Invitrogen, USA), which consists of BODIPY fluorophore (FL)-labeled DQ elastin
conjugate and yields fluorescent fragments upon cleavage by elastase enzyme [31].
Fluorescence signals were recorded every 10 min with Tecan Infinate 200 Pro plate
reader (Tecan Group Ltd., Männedorf, Switzerland; excitation 490 nm, emission 520
nm) for 3 h.

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5.2.4. Rhamnolipid assay.
Rhamnolipid was quantified according to the method of Koch et al [32], with
modifications. A subculture was prepared by directly diluting the overnight culture
1:100 into fresh Minimal Medium (49.3 mM Na2HPO4, 50 mM KH2PO4, 4.8 mM
MgSO4, 7.6 mM (NH4)2SO4, 0.6 mM CaCl2, 25 µM FeSO4, 0.162 µM (NH4)6Mo7O24,
38 µM ZnSO4, 14 µM MnCl2, 1.6 µM CuSO4, 0.86 µM CoCl2, 1.9 µM boric acid, 5.5
µM NiCl2, 6.72 µM EDTA, 0.6% glycerol in 18 MΩ deionized water). The cultures
o
were grown for 24 h at 37 C, with shaking (200 rpm). The final cell density was
measured at 600 nm (OD600) using Tecan Infinate 200 Pro plate reader (Tecan Group
Ltd. Männedorf, Switzerland). Supernatants were collected by ̈centrifuging at 10000
rpm for 10 min and extracted twice with Et2O. Organic fractions were concentrated to
yield white solids which were resuspended in deionized water and 0.19% (w/v)
orcinol in 50% H2SO4 was added. The resulting mixture was incubated at 80 oC for 30
min to give a yellow-orange solution. After cooling to room temperature, the
absorbance was measured at 421 nm and results were normalized with final OD600
values.
5.2.5. Pyocyanin Quantification Assay.
An overnight culture of P. aeruginosa PAO1 was diluted to an OD600 of 0.1 then
diluted 100 times into 25 mL of LB Medium in 250 mL flask. The cultures were
o
grown for 48 h at 37 C, with shaking (200 rpm). The cultures were then centrifuged
for 10 min at 10000 rpm and 8 mL of the supernatants were transferred into new tubes.
Briefly, 8 mL of culture supernatant prepared as described above was extracted with
CHCl3 at a ratio of 4:1, followed by extraction with 1 mL of 0.2M HCl. The
absorbance of the upper red phase was measured using the OD520. The data was
normalized by dividing OD520 reading with the final OD600 values.
5.2.6. GFP reporter strain assays
PAO1-gfp, PAO1-lasB-gfp, PAO1-rhlA-gfp and PAO1-pqsA-gfp bioreporter
strains were kind gifts from Singapore Centre for Environmental Life Sciences
Engineering of Nanyang Technology University. An overnight culture of reporter
strain (grown in LB medium at 37 oC, 200 rpm) was diluted in ABTGC medium to a
final optical density at 600 nm (OD600) of 0.02 (2.5 × 108 CFU/mL). An equal amount

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of the bacterial suspension was added to the wells to reach a final inhibitor
concentration. DMSO control (0.1% final concentration) and a blank control were
o
used. The microtiter plate was incubated at 37 C in Tecan Infinate 200 Pro plate
reader (Tecan Group Ltd., Männedorf, Switzerland) to measure the cell density
(OD600) and GFP fluorescence (excitation at 485 nm, emission at 535 nm) at 15 min
intervals for at least 12 h. The inhibition assay for all test compounds and controls
were done in triplicate. The bacterial strains and plasmids used for respective test are
shown in Table 2.
Table 2
5.2.7. 3D imaging of biofilm development.
To visualize bacterial biofilm formation, the method described previously was
used for biofilm formation but in a glass-bottom 96-well plate (12–556–38, Fisher
Scientific International Inc., Franklin, MA) [33]. After 20 h of incubation at 30 oC, the
supernatant was removed, and the well surface was rinsed briefly with 1 × PBS buffer
three times. PBS buffer (100 ml) containing 1 mM of SYTO 9 and 10 mM of
propidium iodide was added into the well. The plate was incubated for 15 min in the
dark. Fluorescent images were acquired with a confocal microscope (Zeiss LSM800)
to monitor SYTO 9 and propidium iodide (PI) fluorescence. Images were obtained
using an oil immersion × 60 objective lens.
5.2.8. Cytotoxicity Assay.
The cytotoxicity of 6a on RAW 264.7 cells was investigated with an MTT assay.
Cells were seeded into a 96-well plate at a density of 1 × 105 cells/well for 16 h. After
removing the supernatant, the cells were treated with various concentrations of 6a at
sub-MIC concentrations (40, 20, 10, 5, 2.5 and 0 µM) for an incubation lasting 24 h.
MTT solution (0.02 mL) (Sigma Chemical Co.) at concentration of 5 mg/mL was
added and incubated for another 4 h. Finally, supernatant was removed, and 0.2 mL of
dimethyl sulfoxide (DMSO) was added to each well. 10 min of shaking was necessary
before OD570 was measured on a ELISA plate reader.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(81673336), Pearl River S&T Nova Program of Guangzhou (201806010116), and the
Fundamental Research Funds for the Central University (21617478).

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